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 TM

M E THODS IN M OLECULAR B IOLOGY

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

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Nitrogen Fixation
Methods and Protocols

Edited by

Markus W. Ribbe
University of California, Irvine, CA, USA
Editor
Markus W. Ribbe
Department of Molecular Biology &
Biochemistry
University of California
Irvine, California, USA
[email protected]

ISSN 1064-3745 e-ISSN 1940-6029

ISBN 978-1-61779-193-2 e-ISBN 978-1-61779-194-9
DOI 10.1007/978-1-61779-194-9
Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2011931959

© Springer Science+Business Media, LLC 2011

All rights reserved. This work may not be translated or copied in whole or in part without the written permission of
the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013,
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Preface
Nitrogenase is a complex metalloenzyme that catalyzes one of the most remarkable chem-
ical transformations in biological systems: the nucleotide-dependent reduction of atmo-
spheric dinitrogen to bioavailable ammonia (designated biological nitrogen fixation). The
fundamental significance of this process has prompted vigorous research on nitrogenase.
However, few problems in protein biochemistry have proven to be as challenging and
recalcitrant as the molecular description of nitrogenase. Although progress has been made
toward deciphering the enzymatic and biosynthetic mechanisms of this enzyme system,
further development is hampered by the complexity of nitrogenase that makes it impossi-
ble to study this enzyme by any singular method. To overcome this problem, the research
area of nitrogen fixation has evolved into a highly interdisciplinary field that tackles the
remaining questions of nitrogenase mechanism and biogenesis with a combination of
methods. This volume attempts to provide an up-to-date, in-depth overview of the meth-
ods that have been applied to studying the nitrogenase at a molecular level. A large ensem-
ble of approaches is covered in this volume, ranging from genetic, biochemical, spectro-
scopic, and chemical methods to theoretical calculations. In addition, techniques used to
study an enzyme system that is homologous to nitrogenase are described in this book. A
project of this scope requires the timely cooperation of many participants, and I greatly
appreciate the willingness of all authors to face and meet such a demanding schedule. I
hope that this volume, written by recognized experts in their particular areas, will be use-
ful for anyone who is interested in nitrogenase research and who is willing to take charge
of addressing the remaining mechanistic and biosynthetic questions of this fascinating
enzyme system.

Irvine, California, USA Markus W. Ribbe

v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

SECTION I NITROGEN FIXATION

1. Historic Overview of Nitrogenase Research . . . . . . . . . . . . . . . . . . . . 3
Yilin Hu and Markus W. Ribbe
2. Mechanism of Mo-Dependent Nitrogenase . . . . . . . . . . . . . . . . . . . . 9
Zhi-Yong Yang, Karamatullah Danyal, and Lance C. Seefeldt
3. Assembly of Nitrogenase MoFe Protein . . . . . . . . . . . . . . . . . . . . . . 31
Chi-Chung Lee, Aaron W. Fay, Jared A. Wiig, Markus W. Ribbe,
and Yilin Hu
4. Genomic Analysis of Nitrogen Fixation . . . . . . . . . . . . . . . . . . . . . . 49
Ina P. O’Carroll and Patricia C. Dos Santos
5. Enzymatic Systems with Homology to Nitrogenase . . . . . . . . . . . . . . . . 67
Jürgen Moser and Markus J. Bröcker

SECTION II GENETIC AND BIOCHEMICAL METHODS

6. Molecular Biology and Genetic Engineering in Nitrogen Fixation . . . . . . . . . 81
Patricia C. Dos Santos
7. Purification of Nitrogenase Proteins . . . . . . . . . . . . . . . . . . . . . . . . 93
Jared A. Wiig, Chi-Chung Lee, Aaron W. Fay, Yilin Hu, and Markus W. Ribbe
8. Assays of Nitrogenase Reaction Products . . . . . . . . . . . . . . . . . . . . . . 105
William E. Newton and Michael J. Dilworth
9. Methods for Nitrogenase-Like Dark Operative Protochlorophyllide
Oxidoreductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Jürgen Moser and Markus J. Bröcker

SECTION III SPECTROSCOPIC METHODS

10. X-Ray Crystallography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Lauren E. Roth and F. Akif Tezcan
11. X-Ray Absorption Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Serena DeBeer
12. Small Angle X-Ray Scattering Spectroscopy . . . . . . . . . . . . . . . . . . . . 177
David W. Mulder and John W. Peters

vii
viii Contents

13. Electron Paramagnetic Resonance Spectroscopy . . . . . . . . . . . . . . . . . . 191

Karamatullah Danyal, Zhi-Yong Yang, and Lance C. Seefeldt
14. Magnetic Circular Dichroism Spectroscopy . . . . . . . . . . . . . . . . . . . . 207
Brian J. Hales
15. Mössbauer Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Boi Hanh Huynh

SECTION IV CHEMICAL METHODS

16. Protocols for Cofactor Isolation of Nitrogenase . . . . . . . . . . . . . . . . . . 239
Aaron W. Fay, Chi-Chung Lee, Jared A. Wiig, Yilin Hu,
and Markus W. Ribbe
17. Techniques for Functional and Structural Modeling of Nitrogenase . . . . . . . . 249
Patrick L. Holland

SECTION V THEORETICAL CALCULATIONS

18. Nitrogenase Structure and Function Relationships by Density Functional Theory . 267
Travis V. Harris and Robert K. Szilagyi
19. Modeling the MoFe Nitrogenase System with Broken Symmetry Density
Functional Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
Gregory M. Sandala and Louis Noodleman
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Contributors
MARKUS J. BRÖCKER • Institut für Mikrobiologie, Technische Universität Braunschweig,
Braunschweig, Germany
KARAMATULLAH DANYAL • Department of Chemistry and Biochemistry, Utah State
University, Logan, UT, USA
SERENA DEBEER • Department of Chemistry and Chemical Biology, Cornell University,
Ithaca, NY, USA
MICHAEL J. DILWORTH • School of Biological Sciences and Biotechnology, Center for
Rhizobium Studies, Murdoch University, Murdoch, WA, Australia
PATRICIA C. DOS SANTOS • Department of Chemistry, Wake Forest University, Wiston-
Salem, NC, USA
AARON W. FAY • Department of Molecular Biology and Biochemistry, University of
California, Irvine, CA, USA
BRIAN J. HALES • Department of Chemistry, Louisiana State University, Baton Rouge,
LA, USA
TRAVIS V. HARRIS • Department of Chemistry and Biochemistry, Astrobiology Biogeo-
chemistry Research Center, Montana State University, Bozeman, MT, USA
PATRICK L. HOLLAND • Department of Chemistry, University of Rochester, Rochester,
NY, USA
YILIN HU • Department of Molecular Biology and Biochemistry, University of California,
Irvine, CA, USA
BOI HANH HUYNH • Department of Physics, Emory University, Atlanta, GA, USA
CHI-CHUNG LEE • Department of Molecular Biology and Biochemistry, University of
California, Irvine, CA, USA
JÜRGEN MOSER • Institut für Mikrobiologie, Technische Universität Braunschweig,
Braunschweig, Germany
DAVID W. MULDER • Department of Chemistry and Biochemistry, Astrobiology Biogeo-
catalysis Research Center, Montana State University, Bozeman, MT, USA
WILLIAM E. NEWTON • Department of Biochemistry, Virginia Polytechnic Institute &
State University, Blacksburg, VA, USA
LOUIS NOODLEMAN • Department of Molecular Biology, The Scripps Research Institute,
La Jolla, CA, USA
INA P. O’CARROLL • HIV Drug Resistance Program, National Cancer Institute at
Frederick, Frederick, MD, USA
JOHN W. PETERS • Department of Chemistry and Biochemistry, Astrobiology Biogeocatal-
ysis Research Center, Montana State University, Bozeman, MT, USA
MARKUS W. RIBBE • Department of Molecular Biology and Biochemistry, University of
California, Irvine, CA, USA
LAUREN E. ROTH • Department of Chemistry and Biochemistry, University of California,
San Diego, La Jolla, CA, USA
GREGORY M. SANDALA • Department of Molecular Biology, The Scripps Research Insti-
tute, La Jolla, CA, USA

ix
x Contributors

LANCE C. SEEFELDT • Department of Chemistry and Biochemistry, Utah State Univer-

sity, Logan, UT, USA
ROBERT K. SZILAGYI • Department of Chemistry and Biochemistry, Astrobiology Biogeo-
chemistry Research Center, Montana State University, Bozeman, MT, USA
F. AKIF TEZCAN • Department of Chemistry and Biochemistry, University of California,
San Diego, La Jolla, CA, USA
JARED A. WIIG • Department of Molecular Biology and Biochemistry, University of
California, Irvine, CA, USA
ZHI-YONG YANG • Department of Chemistry and Biochemistry, Utah State University,
Logan, UT, USA
 Section I

Nitrogen Fixation
 Chapter 1

Historic Overview of Nitrogenase Research

Yilin Hu and Markus W. Ribbe

Abstract
The history of nitrogenase research dates all the way back to the 1800s. This chapter provides a brief
account of the advances in this particular research area over the past few hundred years, which include
such events as the initial discovery of biological nitrogen fixation, the preparation of active cell-free
extracts, the purification of nitrogenase enzyme, the proposal of the Thorneley–Lowe model, and the
report of x-ray crystallographic structures of the component proteins of nitrogenase.

Key words: Biological nitrogen fixation, nitrogenase, cell-free extracts, purification, x-ray
crystallography.

1. The Discovery
of Biological
Nitrogen Fixation
The year 1888 represents one of the most important years in
in 1888 the history of biological nitrogen fixation. In this year, Hellriegel
and Wilfarth demonstrated conclusively that the legumes could
use atmospheric nitrogen (N2 ) for growth and that the fixation
of nitrogen into the plant depended on bacteria that could be
found in its root nodules (1). Despite the fact that there had
been many claims before about the existence of biological nitro-
gen fixation, this report was the first in which this process was
proven to exist without any doubt. In the same year, Beijerinck
isolated the responsible bacteria from the root nodules (2) and
the combination of these reports formed the foundation for text-
books on nitrogen fixation for decades to come. Several years
later, the free-living nitrogen-fixing soil-bacteria Clostridium pas-
teurianum, Azotobacter chroococcum, and A. agilis were discov-
ered (3, 4), and these discoveries resulted in attempts to establish

M.W. Ribbe (ed.), Nitrogen Fixation, Methods in Molecular Biology 766,

DOI 10.1007/978-1-61779-194-9_1, © Springer Science+Business Media, LLC 2011

3
4 Hu and Ribbe

research programs on this topic in many countries. On the other

hand, although these organisms attracted a great deal of atten-
tion at this time, only few detailed studies on their physiology
and metabolic reactions were reported. This was mainly due to
the fact that techniques available at the time were not adequate to
tackle the scientific problems in the field, ultimately forcing many
scientists to leave this research area to perform easier experiments
elsewhere. One major methodical barrier was the availability of a
reliable method for the determination of nitrogen. The Kjeldahl
assay was the only tool available to early scientists for nitrogen
quantification; unfortunately, this method is not very sensitive,
nor is it very accurate. It was apparent that the field was in a dire
need for new techniques in order to revitalize the interest and
ensure further progress in this scientific topic.

2. Major
Advances Before
1960
A paper published in the year 1928 by Meyerhof and Burk (5)
led to a revival of the interest in the chemistry and biology of
nitrogen fixation. This was due to (i) the introduction of the
use of a microrespirometer as a technique for the indirect mea-
surement of fixed nitrogen and (ii) the development of concepts
that allowed a detailed examination of the properties of a specific
enzyme system in growing cells. Two years later, Bortels reported
the dependency of the nitrogenase of A. chroococcum on molybde-
num (6). In 1942, Burris presented the first conclusive evidence
that ammonia—the compound terminating the inorganic phase
of biological nitrogen fixation—might be the key intermediate
of this process that was eventually incorporated into the cell pro-
teins (7). In the meantime, despite the gradual recognition of cer-
tain fundamental biochemical properties of nitrogenase, further
investigations of this enzyme system would still require a major
methodological breakthrough.

3. The Successful
Preparation of
Nitrogen-Fixing
The first cell-free enzyme preparation that had the consistent
Cell-Free Enzyme ability to fix N2 was reported in 1960 by Carnahan and his
Extracts in 1960 colleagues (8). This report marked the arguably most impor-
tant breakthrough in the research field of nitrogen fixation and
paved the way for meaningful investigations of the enzyme system
 Historic Overview of Nitrogenase Research 5

required for nitrogen fixation on a molecular level. The secret to

the success of Carnahan and his colleagues’ work was the exclu-
sion of air from their preparations of active cell-free extracts from
C. pasteurianum. Following this work, the preparation of active
cell-free extracts from several other anaerobic organisms, such as
Klebsiella pneumoniae, was reported within a month (9). How-
ever, the preparation of active extracts from an obligate, aerobic
organism has remained unaccomplished for several more years.
This task was eventually accomplished in 1964 by Bulen and col-
leagues, when they successfully prepared active cell-free extracts
from A. vinelandii (10). The same group of researchers imme-
diately followed their success by another discovery that sodium
dithionite could be used as a nearly universal electron donor in
studies of the nitrogenase enzyme system (11). Furthermore, they
identified the last missing pieces of the puzzle for a highly active
cell-free extract of nitrogenase: ATP and ATP-regenerating sys-
tem (comprising creatine phosphate, and creatine kinase). Addi-
tion of these compounds served to (i) regenerate ATP from ADP,
thus providing a constant supply of energy source for the enzy-
matic activity; and (ii) remove ADP from the reaction, which was
an inhibitor for the enzyme system.

4. The
Identification
of the Binary
The discovery of the key parameters for the reproducible
Nature of
preparation of nitrogen-fixing cell-free enzyme extracts laid the
Nitrogenase foundation for the subsequent success in the purification of
After 1960 nitrogenase enzyme. Between 1966 and 1967, Bulen, LeComte,
Mortenson, and colleagues reported that nitrogenases of both A.
vinelandii and C. pasteurianum were composed of two brown
proteins and that both protein components were required for
the enzymatic activity of nitrogenase (12, 13). The two nitroge-
nases from A. vinelandii and C. pasteurianum were very similar
in properties: one component showed a relatively higher molec-
ular mass (200,000–250,000) and contained molybdenum, iron,
and acid-labile sulfur; the other was a smaller protein (50,000–
60,000) and contained only iron and acid-labile sulfur. Both
component proteins were composed of multiple subunits and
could be irreversibly destroyed by air. These nitrogenase pro-
teins were given numerous different names in the follow-up pub-
lications. However, the most commonly used designations are
molybdenum–iron (MoFe) protein or dinitrogenase for the larger
component and iron (Fe) protein or dinitrogenase reductase for
the smaller component.
6 Hu and Ribbe

5. Investigations
of Nitrogenase
on a Molecular
The availability of nitrogenase in the purified state sets the stage
Level Since the for extensive investigations of this enzyme system on a molec-
1990s ular level. In 1985, Lowe and Thorneley proposed a model of
nitrogenase mechanism, which has remained the best description
of nitrogenase reaction to date (14). Between 1992 and 1993,
Rees and coworkers solved the crystal structures of both compo-
nent proteins of nitrogenase from A. vinelandii (15–17). These
structural reports represented one of the biggest accomplishments
in the field since the purification of nitrogenase. Subsequently,
the structurally derived information prompted chemical synthetic
efforts of the metal centers of nitrogenase (18–23) and, together
with the genetically based knowledge (24; also see Chapter 4), it
facilitated mechanistic (25–27; also see Chapter 2) and biosyn-
thetic (28–30; also see Chapter 3) investigations of this unique
enzyme system. Despite the significant advances in the field over
the past decades, the complexity of nitrogenase and the difficulty
in protein handling have made it clear to the community that it
is impossible to obtain a detailed molecular description of nitro-
genase on the basis of a singular approach. Thus, recent efforts
toward addressing the remaining questions in the research area of
nitrogenase often feature collaborations between various groups,
taking full advantage of combined genetic (see Chapter 6), bio-
chemical (see Chapters 7 and 8), spectroscopic (see Chapters
10, 11, 12, 13, 14, and 15), chemical (see Chapters 16 and 17),
and theoretical (see Chapters 18 and 19) methods. Such an inter-
disciplinary approach may prove instrumental in solving the bio-
logical riddle of nitrogen fixation in the near future.

Acknowledgments

The authors are supported by the National Institutes of Health

grant GM 67626 (M.W.R.) and Herman Frasch Foundation
grant 617-HF07 (M.W.R.).

References

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suchungen über die Stickstoffnährung der der Papilionaceen-Knölchen. Bot Ztg
Gramineen und Leguminosen. Beil Z Ver dt 46:725–735; 741–750; 757–771; 781–790;
ZuchInd, 1–234 797–804
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3. Winogradsky S (1893) Sur l’assimilation 18. Lee SC, Holm RH (2003) Speculative
de l’azote gaseux de l’atmosphère par les synthetic chemistry and the nitrogenase
microbes. C r hedb Séanc Acad Sci Paris problem. Proc Natl Acad Sci USA 100:
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4. Beijerinck MW (1901) Über oligoni- 19. Lee SC, Holm RH (2004) The clusters of
trophile Mikroben. ZentBl Bakt ParasitKde nitrogenase: synthetic methodology in the
7:561–582 construction of weak-field clusters. Chem
5. Meyerhof O, Burk D (1928) On the fixation Rev 104:1135–1158
of air nitrogen through Azotobacter. Z Phys 20. Groysman S, Holm RH (2009) Biomimetic
Chem 139:117–142 chemistry of iron, nickel, molybdenum, and
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bei der biologischen Stickstoffbindung. Arch chemistry 48:2310–2320
Mikrobiol 1:333–342 21. Coucouvanis D (1994) Fe/S and Fe/Mo/S
7. Burris H (1942) Distribution of isotopic clusters as speculative models for the metal
nitrogen in Azotobacter vinelandii. J Biol centers in uncommon Fe/S proteins and
Chem 143:509–517 the Fe/Mo protein of the nitrogenases. Adv
8. Carnahan JE, Mortenson LE, Mower HF Inorg Biochem 9:75–122
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free extracts of Clostridium pasteurianum. Tatsumi K (2003) Synthesis of the P-cluster
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extracts of Azotobacter. Biochem Biophys Res Ertesvåg H, Espin G, Rubio LM, Valla S,
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 Chapter 2

Mechanism of Mo-Dependent Nitrogenase

Zhi-Yong Yang, Karamatullah Danyal, and Lance C. Seefeldt

Abstract
Nitrogenase is the enzyme responsible for biological reduction of dinitrogen (N2 ) to ammonia, a form
usable for life. Playing a central role in the global biogeochemical nitrogen cycle, this enzyme has been
the focus of intensive research for over 60 years. This chapter provides an overview of the features of
nitrogenase as a background to the subsequent chapters of this volume that detail the many methods
that have been applied in an attempt to gain a deeper understanding of this complex enzyme.

Key words: Nitrogen fixation, Fe protein, MoFe protein, mechanism, metalloenzyme, MgATP.

1. Nitrogen
Fixation
Dinitrogen (N2 ) is the major constituent (79%) of the Earth’s
atmosphere, representing the largest global pool of nitrogen (N).
While nitrogen is essential to all life, the vast reservoir of dinitro-
gen in the atmosphere is unusable by most organisms (1, 2). This
is largely a consequence of the high bond dissociation energy for
the N2 triple bond (3), making the breaking of this bond and “fix-
ation” of the nitrogen to forms usable to living organisms ener-
getically challenging. Dinitrogen can be fixed with considerable
energy input by addition of electrons and protons to yield two
ammonia (NH3 ) molecules. In the industrial Haber–Bosch pro-
cess for fixing dinitrogen, the reaction is carried out at high tem-
peratures (∼450◦ C) and pressures (>200 atm) in the presence of
an iron catalyst, with the electrons and protons coming from H2
(1, 4–6). This process is extremely energy demanding, utilizing
approximately 1% of the total fossil fuel used globally (7).

M.W. Ribbe (ed.), Nitrogen Fixation, Methods in Molecular Biology 766,

DOI 10.1007/978-1-61779-194-9_2, © Springer Science+Business Media, LLC 2011

9
10 Yang, Danyal, and Seefeldt

The other major pathway for reduction of dinitrogen is

through the action of select microorganisms (called diazotrophs)
that carry out a process called biological nitrogen fixation
(6, 8). The first step in the assimilation of N2 by these organ-
isms is the reduction of N2 to two ammonia molecules catalyzed
by a complex metalloenzyme called nitrogenase (9, 10). Nitroge-
nases occur across a wide range of microbes and sequencing of the
nitrogenase genes (nif genes) reveals considerable sequence diver-
sity among the enzymes (10–12). Despite this sequence diver-
sity, the majority of nitrogenases share some common features.
For example, most enzymes are composed of two component
proteins: a large component having at least an α2 β2 subunit com-
position and a smaller component having a γ2 subunit composi-
tion. All known nitrogenases contain iron–sulfur clusters in both
component proteins. The site of N2 binding and reduction is one
of the iron–sulfur clusters contained in the larger component pro-
tein. This active site metal cluster can contain, in addition to Fe
atoms, a heterometal atom (Mo or V) (9, 10, 13, 14). The best
studied nitrogenase is the Mo-dependent enzyme, which appears
to be the paradigm for nitrogenases (9, 13, 15–21). Given that
most of the mechanistic information known about nitrogenases is
for the Mo-based enzyme, this chapter will focus on this enzyme.
Other nitrogenases, sometimes called alternative nitrogenases, are
reviewed elsewhere (10, 12).

2. Mo-Dependent
Nitrogenase:
Overview
The two component proteins of the Mo-dependent nitrogenase
are called the iron (Fe) protein (or dinitrogenase reductase)
and the molybdenum–iron (MoFe) protein (or dinitrogenase)
(Fig. 2.1). These two component proteins work together to cat-
alyze the reduction of dinitrogen in a complex reaction with an
ideal reaction stoichiometry shown as follows (22):

N2 + 8e − + 16MgATP + 8H+ → 2NH3 + H2 + 16MgADP + 16Pi [1]

A breakthrough in understanding nitrogenase came from the

X-ray crystal structures of the component proteins solved
individually (23–42) and when bound together (43–46).
The Fe protein, the only known reductant of the MoFe
protein that can support substrate reduction, is a homodimer
that contains two nucleotide (MgATP or MgADP) binding sites,
one on each subunit, and a single [4Fe–4S] cluster that bridges
the two subunits (Fig. 2.1) (25). The MoFe protein is an α2 β2
 Mechanism of Mo-Dependent Nitrogenase 11

Fig. 2.1. Crystal structure of the Fe and MoFe protein components of Mo-dependent
nitrogenase showing the nucleotides, metal clusters, and electron transfer pathways.
(Left) Cartoon representation of MoFe protein (pdb code: 1M1N) with the α-subunits and
the β-subunits and Fe protein (pdb code: 1FP6) with the γ-subunits. (Right) Structures
of MgADP and the three metalloclusters of nitrogenase. The figure was generated using
the computer program PyMol.

heterotetramer. Each αβ dimeric unit contains two unique met-

alloclusters: a P-cluster ([8Fe–7S]) and an FeMo cofactor ([7Fe-
9S-Mo-X-(R)-homocitrate]) (Fig. 2.1) (27, 28, 47). Each αβ-
unit appears to function as a catalytic unit independent of the
other αβ pair. During the catalytic cycle, an Fe protein binds tran-
siently to one MoFe protein αβ unit. During this encounter, one
electron is transferred from the [4Fe–4S] cluster of the Fe pro-
tein to the MoFe protein. This electron transfer step is coupled to
the hydrolysis of a minimum of two MgATP molecules (16, 48).
Following electron transfer and ATP hydrolysis, the Fe protein
disengages from the MoFe protein and a new Fe protein binds
in its place to repeat the cycle. Given that only one electron is
transferred per cycle, a minimum of eight encounters must occur
to reduce N2 (equation [1]).

2.1. Fe Protein The Fe protein is a homodimer (coded for by the nifH gene)
with a molecular mass of approximately 64,000 Da (25, 49). In
addition to delivering electrons to the MoFe protein, the Fe pro-
tein also is known to function in the maturation of the MoFe
12 Yang, Danyal, and Seefeldt

protein and in the bioassembly of the active site metal cluster

called FeMo cofactor (49, 50). This maturation role for the Fe
protein does not appear to require electron transfer or the ATP
hydrolysis function (9) and is covered in another chapter of this
volume.

2.1.1. Redox Properties The Fe protein contains a single [4Fe–4S] cluster that serves
of the [4Fe–4S] Cluster as a carrier of electrons. The X-ray structure of the Fe protein
revealed that this cluster is symmetrically ligated between the two
Fe protein subunits, with each subunit contributing two cysteine
ligands (Fig. 2.1). The [4Fe–4S] is known to access three redox
states (9, 21):

Em = −300mV Em = −790 mV
⎯⎯⎯⎯⎯
→[4Fe−4S]1+←⎯⎯⎯⎯
[4Fe−4S]2+←⎯⎯⎯⎯
⎯ ⎯⎯⎯⎯⎯
→[4Fe−4S]0
⎯ [2]

The [4Fe–4S]2+/1+ redox couple is operational during substrate

reduction supported by reductants such as dithionite or ferre-
doxin (9, 51, 52). A more reduced (0) oxidation state (termed
the “all-ferrous” state) can be achieved in vitro by incubating the
Fe protein with reductants with very negative potentials (e.g., Ti-
citrate) (53–57). It has been demonstrated that the all-ferrous
state can participate in the delivery of electrons to the MoFe pro-
tein when strong reductants are used (58). However, the role of
this all-ferrous state during catalysis in vivo remains unknown.
The 1+ oxidation state (FeRed ) of the [4Fe–4S] cluster is the
dominant state in the as-purified enzyme in the presence of the
reductant dithionite (S2 O2−4 ) (59–61). This oxidation state of the
Fe protein is paramagnetic, with the four Fe atoms distributed as
3Fe2+ and 1Fe3+ . This state gives rise to an EPR spectrum at low
temperatures (∼5 K) that has been assigned as a mixture of two
spin states (S = 1/2 and S = 3/2 spin). This mixed spin state has
been confirmed in the Mössbauer and MCD spectra. The ratio of
the two spin states can be shifted by addition of other reagents
such as urea or glycerol into the sample solution (61).
The 1+ oxidation state of the [4Fe–4S] cluster can be
reversibly oxidized by the removal of one electron, achieving the
2+ oxidation state (FeOx ), with the iron atoms distributed as
2Fe2+ and 2Fe3+ (54, 61, 62). This oxidation state of the [4Fe–
4S] cluster is diamagnetic and therefore is EPR silent. The oxi-
dation of the [4Fe–4S] cluster from the 1+ to the 2+ oxidation
state can be achieved by the treatment of the Fe protein with
redox-active dyes of sufficiently positive potential (e.g., thionine,
methylene blue, and indigo disulfonate) (54, 63), whereas reduc-
tion from the 2+ to the 1+ oxidation state can be achieved by
the addition of a number of reductants (e.g., dithionite) (64).
This reversibility allows the establishment of the midpoint reduc-
tion potential (Em ) for the [4Fe–4S]2+/1+ redox couple using
 Mechanism of Mo-Dependent Nitrogenase 13

voltametric and coulometric methods (65–67). The values of Em

are dependent on the organism from which the Fe protein is puri-
fied and the presence or absence of bound nucleotides (9). The
Em for the [4Fe–4S]2+/1+ couple of the Fe protein from Azoto-
bacter vinelandii is measured to be –300 mV in the absence of
nucleotides (equation [2]) (62, 68). When MgATP is added to
the protein, the Em value shifts more negative to –430 mV (68).
MgADP shifts the Em to –440 mV (68).
It is well established from kinetic and spectroscopic studies
that the [4Fe–4S]2+/1+ redox couple of the [4Fe–4S] cluster in
the Fe protein is functional during the catalytic cycle of nitroge-
nase (9, 16, 21). While the Fe protein in the 1+ oxidation state
is bound to the MoFe protein, an electron is transferred from
the Fe protein to the MoFe protein, resulting in oxidation of
the [4Fe–4S] cluster to the 2+ oxidation state. The consensus
model requires the oxidized Fe protein (2+) to dissociate from
the MoFe protein and for the [4Fe–4S] cluster to be reduced
back to the 1+ oxidation state by a reduced electron carrier pro-
tein (e.g., flavodoxin or ferredoxin) (51, 52), thereby readying
the Fe protein for another round of electron transfer to the MoFe
protein.

2.1.2. The Fe Protein Early work on nitrogenase revealed that the Fe protein could bind
Binds Nucleotides nucleotides and that the hydrolysis of nucleotides by the nitroge-
nase complex was critical to the transfer of an electron from the Fe
protein to the MoFe protein (16, 69). The Fe protein binds two
nucleotides, one to each subunit. The nucleotide binding sites on
the Fe protein are on the opposite end of the Fe protein from
the [4Fe–4S] cluster (30, 31, 37, 38, 41, 43–46). The dissoci-
ation constants (Kd ) for nucleotide binding to Fe protein have
been determined by a number of techniques (70). Recent studies
using isothermal titration calorimetry have supported earlier stud-
ies showing that the redox state of the [4Fe–4S] cluster impacts
the affinity for nucleotide binding (71). The 2+ oxidation state
binds MgATP with the highest affinity (Kd = 45 μM), while the
1+ oxidation state has a lower affinity (Kd = 500 μM) for this
nucleotide. Further, these studies have revealed positive coopera-
tivity in the binding of the two nucleotides to both the reduced
and oxidized states of the Fe protein (72). For the reduced state
(FeRed ) the Kd values for the binding of the first and second
MgATP molecules are reported to be Kd1 = 500 μM and Kd2 =
170 μM, respectively. A divalent metal is required for the binding
of nucleotides to the Fe protein. While a number of different met-
als will work, it is thought that Mg2+ is the physiologically rele-
vant metal (73). The Fe protein can also bind other nucleotide tri-
and di-phosphates (e.g., GTP and GDP) with reasonable affinity,
although it is widely believed that ATP and ADP are the relevant
nucleotides in vivo (74).
14 Yang, Danyal, and Seefeldt

While the Fe protein binds MgATP, it shows undetectable

rates of hydrolysis in the absence of the MoFe protein (21).
It is only after the Fe protein binds to the MoFe protein that
hydrolysis is activated. This observation has been explained from
examination of X-ray structures of the Fe protein with bound
nucleotides as the movement of a likely catalytic base into place
to activate hydrolysis following Fe protein binding to the MoFe
protein. A detailed understanding of the specific interactions of
nucleotides with the Fe protein has been achieved from the
X-ray structures of Fe proteins in various nucleotide-bound states
(43–46).

2.1.3. Nucleotide There is ample evidence showing that the binding of nucleotides
Binding Induces Protein to the Fe protein induces conformational changes to the protein
Conformational Changes structure that impact many aspects of its function. For exam-
in the Fe Protein ple, the binding of MgATP or MgADP to the Fe protein shifts
the Em for the [4Fe–4S]2+/1+ redox couple to more negative
potentials by about –120 mV (described above). Many other
methods also reveal that nucleotides change the properties of the
[4Fe–4S] cluster (16, 21). What is clear is that these changes are
not the result of nucleotides binding directly to the [4Fe–4S]
cluster, but rather a result of nucleotide-induced protein con-
formational changes impacting the cluster over a distance. The
nucleotide binding sites are located approximately 15 Å away
from the [4Fe–4S] cluster (30, 31, 37, 38, 41, 43–46). While
a number of studies report changes in the electronic properties
of the [4Fe–4S] cluster as a result of nucleotides binding to the
Fe protein, it is more recent studies using both small angle X-ray
scattering (SAXS) (75) and X-ray crystallography that are provid-
ing clearer pictures of the larger structural changes induced in the
Fe protein upon nucleotide binding. These are reviewed in other
chapters of this volume.

2.2. MoFe Protein The MoFe protein is an α2 β2 heterotetramer (Mr ∼ 250,000 Da)
with the α and β subunits encoded by the nifD and nifK
genes, respectively (9, 49). Each αβ-dimeric catalytic unit contains
one active site metallocluster, the FeMo cofactor ([7Fe-9S-Mo-
X-(R)-homocitrate]) (76) and one electron carrier cluster, the
P-cluster ([8Fe–7S]). The X-ray crystal structure of the MoFe
protein reveals that the FeMo cofactor is embedded solely in the
α-subunit, while the P-cluster is located at the interface between
the α and β subunits (23, 24). Several structures of the complex
of the Fe protein bound to the MoFe protein reveal the interfaces
where the Fe protein and MoFe protein dock (43–46). These
structures place the P-cluster directly in-line and between the
Fe protein [4Fe–4S] cluster and the FeMo cofactor (Fig. 2.1).
The distance between the [4Fe–4S] cluster and the P-cluster
varies depending on the nucleotide-bound state of the Fe protein,
 Mechanism of Mo-Dependent Nitrogenase 15

Fig. 2.2. Structure of P-cluster in oxidized and reduced states. The reduced state (PN ) of the P-cluster (a) and the oxidized
state (POX ) of the P-cluster (b) are shown (3MIN.pdb and 2MIN.pdb, respectively).

leading to a model wherein one role of nucleotides is to alter this

electron transfer distance and therefore the electron transfer rate.
The arrangement of the three metalloclusters suggests an elec-
tron transfer chain from the [4Fe–4S] cluster to the P-cluster and
finally to the FeMo-cofactor active site (46).

2.2.1. P-Clusters Early Mössbauer spectroscopic studies of the MoFe protein

revealed that the P-cluster was composed of eight ferrous Fe
atoms in the resting state in the presence of dithionite (termed
the PN state) (77). The X-ray structures revealed the nature of
this unusual cluster (Fig. 2.2) as being composed of two cubic
[4Fe–4S] subclusters sharing a common sulfide ligand at one cor-
ner (23, 24, 27, 33, 42). Each Fe atom is coordinated by two
or three sulfide ligands and one terminal or bridging cysteinyl
ligand from a cysteine residue in the α or β subunit. Upon oxi-
dation of the MoFe protein, the P-cluster is oxidized by one or
two electrons, which results in significant structural rearrange-
ment (33, 42). Upon oxidation, one of the cubic units is opened
up with two Fe–S bonds (Fe5–S1 and Fe6–S1) being cleaved and
two novel Fe6–O and Fe5–N bonds being formed. Further, a
serinate-O (β-188Ser ) and a backbone amide-N (α-88Cys ) become
ligands to Fe atoms (Fig. 2.2) (42).
From in vitro studies using dye oxidants, it has been shown
that the resting state of the P-cluster (PN ) can be oxidized by up
to three electrons (P1+ , P2+ , and P3+ ) (equation [3]) (78–81).
The Em values measured for these redox couples are shown for
the A. vinelandii MoFe protein as follows (62, 82, 83):

? −309 −309 +90 Em (mV)

PRed PN P1+ P2+ P3+
[3]

The P1+ and P2+ oxidation states are often collectively referred
to as the POx oxidation state, because both oxidation states are
16 Yang, Danyal, and Seefeldt

usually populated in oxidized states of the MoFe protein. The

P3+ oxidation state is not reversible and so is not believed to be
functioning during catalysis. More reduced states of the P-cluster
from the PN state have not been observed and seem unlikely given
that such a reduction would require reducing Fe atoms beyond
the ferrous oxidation state. The Em for P2+/1+ redox couple is
pH-dependent, shifting Em by –53 mV per increase of one pH
unit (84). The relevance of this pH dependence is not known,
but would be consistent with the coupling of proton and electron
transfer reactions involving this cluster.

2.2.2. FeMo The FeMo cofactor (76), also called the M-cluster, is embedded in
Cofactor—The Active each α subunit of the MoFe protein. The structure of FeMo cofac-
Site of Nitrogenase tor was resolved when the X-ray structure of the MoFe protein
was solved (47). The structure revealed a heterometallocluster
with a composition [7Fe-9S-Mo-X-(R)-homocitrate] (Fig. 2.3)
(28). The cluster is ligated to the peptide matrix through one
cysteine ligand (α-275Cys ) bound to the Fe atom at one end and
through one histidine ligand (α-442His ) bound to the Mo atom
at the other end. The six Fe atoms in the middle part are arranged
as a prismatic structure with each Fe atom coordinated by three
sulfide atoms. Recent high-resolution structures of the MoFe pro-
tein have revealed the presence of a light atom (C, N, or O) at
the center of the Fe cage of unknown identity (designated as X)
that is presumed to be bound to each of the central six Fe atoms
(28). Homocitrate provides two oxygen atom (C1 carboxylate
and C3 hydroxylate) ligands to the Mo (85). Thus, the overall
structure of the FeMo cofactor can be viewed as one [4Fe-3S-X]

Fig. 2.3. Structure of FeMo cofactor and some of the amino acids surrounding it.
Numbering of atoms uses the system in the original structure. Colors for atoms are
Fe in rust, Mo in magenta, S in yellow, C in dark gray, N in blue, and O in red (online
version only). The figure is generated from PDB file 1M1N.
 Mechanism of Mo-Dependent Nitrogenase 17

cubane and one [Mo-3Fe-3S-X] cubane that are connected by

three bridging sulfides with one shared μ6 -X atom at the center.
Identifying the central atom X has proven difficult (69).
ENDOR studies have suggested that it is not an exchangeable N
atom, but have left open the possibility that it is a non-exchanging
N atom (86–88). A vibrational spectroscopy technique (nuclear
resonance vibrational spectroscopy or NRVS) supports the pres-
ence of a light atom at the center of the Fe cage, but does not
resolve the identity of X (89). Likewise, a number of calcula-
tions support the presence of X, but do not provide a definitive
assignment for X (90–93). To date, there remains no experimen-
tal evidence showing the involvement of X in the catalytic cycle of
nitrogenase. Obviously, understanding X and its roles in catalysis
remains a significant challenge for the field.
FeMo cofactor can be reversibly oxidized or reduced from its
resting state. The resting state of FeMo cofactor (MN ) occurs in
the MoFe protein isolated in the presence of dithionite. This state
is paramagnetic with a rhombic S = 3/2 spin EPR signal (94).
Treatment of the MoFe protein with oxidizing dyes results in the
one-electron oxidation to the MOx state (77, 78). The MOx state
is diamagnetic (S = 0) and EPR silent. The Em for the MOx/N
redox couple is about –40 mV (95). The MN state can be reduced.
Incubation with the Fe protein in the presence of MgATP and
dithionite results in the reduction of FeMo cofactor to an MR
state with an integer spin (S > 1) state that is EPR silent (96, 97).
The Em for the MN/R redox couple has not been measured,
but has been estimated as –465 mV (98). The oxidation states
of the Fe atoms and the Mo atom in the resting state of FeMo
cofactor (MN ) have been examined by Mössbauer and ENDOR
spectroscopies. The Mössbauer study suggested an assignment of
[Mo4+ , 3Fe3+ , 4Fe2+ , 9S2– ] (97), which is supported by cal-
culations using a model with the interstitial X atom (99). The
57 Fe ENDOR study suggested an assignment of [Mo4+ , 1Fe3+ ,

6Fe2+ , 9S2– ] for the resting state FeMo cofactor (100). This later
assignment is consistent with the result from calculations on the
FeMo cofactor without an interstitial X atom (90). FeMo cofac-
tor must accept multiple electrons (two or more) to complete the
reduction of substrates. How these electrons are accumulated on
FeMo cofactor is not known. Possible distribution between the
P-clusters, FeMo cofactor, and bound intermediates remains to
be established.
FeMo cofactor can be extracted from the MoFe protein into
organic solvents (76, 101). A number of studies have been con-
ducted on such extracted FeMo cofactor. While some properties
of the cofactor in solvent are similar to those for the cofactor in
the protein (e.g., EPR spectrum), others are quite different. For
example, the reactivity of FeMo cofactor in solvent is different
from that of FeMo cofactor bound to the protein (e.g., substrate
reduction ability) (102).
18 Yang, Danyal, and Seefeldt

A number of studies support FeMo cofactor as the site of

substrate (and inhibitor) binding (19, 101), although the precise
location of substrate binding is still being pursued (17–19). There
are many possibilities for the substrate binding site: (1) the Mo
atom; (2) one or more of the central Fe atom(s); and (3) some
combination of Fe, S, and Mo atoms. Using spectroscopic meth-
ods such as ENDOR, there is growing evidence for binding of
hydrides (103), alkynes (104, 105), and nitrogenous compounds
(106–110) to one or more of the Fe atoms in the central por-
tion of FeMo cofactor (Fig. 2.3). As yet, no experimental results
have illustrated binding of any substrate or intermediate to Mo,
although this possibility has not been ruled out.
An important tool to gaining insights into substrate bind-
ing to FeMo cofactor has been substitution of amino acids in
the MoFe protein using site-directed mutagenesis. An early study
examined the roles of α-195His in nitrogenase catalysis (Fig. 2.3)
(111–115). Substitution of the α-195His residue by glutamine
resulted in a variant of the MoFe protein that cannot effectively
reduce N2 or azide (N− 3 ), but which retained full rates of reduc-
tion of acetylene and protons. From these studies, it was con-
cluded that α-195His might participate in delivery of protons dur-
ing reduction of nitrogen-containing substrates. Slowing down
proton delivery by substituting for α-195His has been exploited
to trap presumed intermediate states during the reduction of a

Fig. 2.4. Some substrates for nitrogenase.

 Mechanism of Mo-Dependent Nitrogenase 19

number of substrates including hydrazine (N2 H4 ) (106, 107),

diazene (HN = NH) (109), and methyldiazene (MeN = NH)
(107, 108). It is clear that α-195His is not the sole source of pro-
tons for substrate reduction as the rates of reduction of other sub-
strates remain undisturbed when this residue is substituted (115).
Recent work with MoFe proteins containing amino acid sub-
stitutions are providing solid evidence for the site of substrate
binding on FeMo cofactor. These studies have recently been
reviewed (13, 18, 19). While the physiological substrates for
nitrogenase are N2 and protons, a number of other small, multi-
ple bonded compounds have been demonstrated to be substrates.
These have been extensively reviewed elsewhere (9, 19, 21, 116).
Several of the substrates are shown in Fig. 2.4.

3. Nitrogenase
Mechanism
3.1. Fe Protein–MoFe An essential step in the nitrogenase mechanism occurs when the
Protein Complex Fe protein, with two bound MgATP molecules, associates with
Formation the MoFe protein. This associated complex is fleeting, existing
for about 1 s during normal substrate reduction (9). Several
events occur while the two proteins are associated, including the
hydrolysis of the two MgATP molecules to two MgADP and two
Pi molecules and the transfer of one electron from the Fe pro-
tein to the MoFe protein. The order of these two events has not
been definitively established and is the subject of current studies
(19, 20).
The associated state of the nitrogenase complex has been
trapped by a number of different approaches. Five different
types of stable complexes that have been examined include the
following:
(1) A chemical cross-linked complex using a bifunc-
tional chemical reagent 1-ethyl-3-[3-(dimethylamino)
propyl]carbodiimide (EDC) (45, 117, 118).
(2) A non-dissociating complex formed between the Fe pro-
tein from Clostridium pasteurianum and the MoFe protein
from A. vinelandii (119–122).
(3) A non-dissociating complex formed between an Fe pro-
tein with an amino acid deletion (127Leu ) and the MoFe
protein that appears to mimic the ATP-bound state in the
absence of ATP (44, 123).
(4) A non-dissociating complex formed when ADP and AlF− 4
(or BeF− 3 ) are added to the Fe protein and MoFe protein.
In this case, the AlF−
4 appears to be mimicking the depart-
ing phosphate following ATP hydrolysis (43, 124–127).
20 Yang, Danyal, and Seefeldt

(5) Relatively stable complexes of the MoFe protein and the Fe

protein with MgADP or β,γ-methylene MgATP bound or
without nucleotide bound (46).
Analysis of these tight complexes by a number of different
approaches, including X-ray crystallography, is providing insights
into the nitrogenase complex such as the following:
(1) AlF− 4 occupies the binding site where the γ-phosphate por-
tion of MgATP was expected to be (43, 46).
(2) The subunits of Fe protein are considerably reoriented
during complex formation and nucleotide hydrolysis. The
movement of two segments of amino acids in the Fe pro-
tein (called switches I and II) appears to connect to the
[4Fe–4S] cluster, possibly controlling the nucleotide-
induced changes in the properties of the [4Fe–4S] cluster
(44, 46).
(3) There are several distinct and mutually exclusive inter-
action sites on the MoFe protein surface that are selec-
tively populated, depending on the Fe protein nucleotide
state (46).
(4) The distance between the Fe protein [4Fe–4S] cluster and
the MoFe protein P-cluster changes by up to 5 Å depend-
ing on the nucleotide bound to the Fe protein (46).
(5) The Em value of the [4Fe–4S] cluster and the P-cluster is
shifted more negative when the two proteins are associated,
favoring electron transfer from the Fe protein to the FeMo
cofactor (123, 128).

3.2. Fe Protein Cycle The Fe protein, being an ATP-dependent reductase of the MoFe
protein, can be thought of as proceeding through a cycle during
its function in the overall nitrogenase catalytic cycle (129, 130).
This Fe protein cycle is summarized in Fig. 2.5. During nitroge-
nase catalysis, the reduced Fe protein, with the [4Fe–4S] cluster
in its 1+ oxidation state, binds two MgATP molecules. The Fe
protein in this state then associates with the MoFe protein (131).
Within this complex, MgATP hydrolysis is activated and electron
transfer occurs, followed by the dissociation of the two proteins.
The spent Fe protein is reactivated by replacing MgADP with
MgATP and reducing the 2+ oxidation state to the 1+ oxidation
state (Fig. 2.5).
While the general features of the Fe protein cycle are known,
several important details remain to be resolved. For example, how
does complex formation activate MgATP hydrolysis and elec-
tron transfer? Which comes first, electron transfer or nucleotide
hydrolysis? How specifically is the energy from nucleotide hydrol-
ysis used in the nitrogenase reaction? What specifically is the role
of the P-cluster in brokering electrons between the Fe protein
 Mechanism of Mo-Dependent Nitrogenase 21

Fig. 2.5. Fe–protein cycle showing the oxidation state changes and MgATP hydrolysis.
Abbreviations used are FePOx for oxidized Fe protein, FePRed for reduced Fe protein,
MoFeP for the oxidation state of MoFe protein before reduction, and MoFePRed for one
more electron reduced state of MoFe protein.

and the active site FeMo cofactor? These and many other ques-
tions need to be addressed in the coming years.

3.3. MoFe–Protein The MoFe protein must accumulate multiple electrons in order to
Cycle achieve the reduction of bound substrates. The details of where
and how these electrons are accumulated in the MoFe protein
are not known (20). A simple notation to designate how many
electrons have been transferred into the resting MoFe protein
(designated as E0 ) is helpful (called the Lowe–Thornely model)
(64, 132–134). This model does not differentiate electrons on
the P-cluster from electrons on the FeMo cofactor (Fig. 2.6),
but rather simply notes the number of electrons accumulated in
the MoFe protein as E1 , E2 , etc., with the subscript indicating the
number of electrons.
The results of a number of kinetic studies have allowed
construction of a MoFe-protein cycle as shown in Fig. 2.6. As

Fig. 2.6. Modified Lowe–Thorneley kinetic scheme for reduction of N2 . In this scheme,
the En represents one functional αβ dimeric unit, which has been reduced by n electrons
relative to the resting state E0 .
22 Yang, Danyal, and Seefeldt

noted in the cycle, there is good evidence indicating that dif-

ferent substrates bind to different reduction states (En ) of the
MoFe protein. Dinitrogen is modeled to bind to E3 or E4 states
(64), which is accompanied by the release of one equivalent of H2
(22, 135). In the absence of N2 , the less reduced E1 and E2 states
are achieved, which are sufficient for proton binding and reduc-
tion to H2 . The non-physiological substrate acetylene is mod-
eled to bind to the E2 state for reduction to ethylene. The bind-
ing of different substrates to different redox states of the MoFe
protein can result in confusing inhibition patterns. For exam-
ple, the inhibition of N2 reduction by acetylene appears to be
non-competitive, while the inhibition of acetylene reduction by
N2 appears to be competitive (136). This apparent contradiction
can be explained by the fact that acetylene binds to the E2 state
whereas N2 binds to more reduced states (64, 137). Thus, acety-
lene appears to be a non-competitive inhibitor of N2 reduction
and N2 a competitive inhibitor of acetylene reduction.
Insights into where and to what states of FeMo-cofactor sub-
strates and inhibitors bind have come in recent years from the
characterization of freeze-trapped MoFe protein during turnover
with substrates or inhibitors. The inhibitor carbon monoxide
(CO) has been extensively characterized trapped to FeMo cofac-
tor (100, 138–142). Application of a variety of spectroscopic
methods to this trapped state has revealed that at low CO con-
centrations, a single CO is likely bound bridging between two Fe
atoms. At high CO concentrations, two CO molecules are pro-
posed to be bound.
Amino acid substitutions in the MoFe protein have been
used, along with freeze trapping to capture a number of different
substrates bound as intermediates to FeMo cofactor. The MoFe
protein variant, the substrate, and the g values of the EPR spec-
trum observed are summarized in Table 2.1. How these states
are trapped is discussed in more detail in another chapter of this
volume.
Some of the key findings from characterization of these
trapped states include the following:
(1) A hydride-trapped state is consistent with two hydrides
bound to Fe atoms in FeMo cofactor (106).
(2) The substrate propargyl alcohol and other alkyne substrates
have been trapped bound side-on to one or more Fe atoms
(104, 105, 143).
(3) Intermediates have been trapped starting from the nitroge-
nous substrates hydrazine, diazene, methyldiazene, and N2 .
These intermediates appear to be bound end-on to Fe
atom(s) (106–110).
(4) A specific Fe atom in the central portion of FeMo cofactor
has been identified as the likely site of binding of several
substrates tested so far (110, 143).
 Mechanism of Mo-Dependent Nitrogenase 23

Table 2.1
Important variants of MoFe protein and EPR parameters of the resulting intermedi-
ates with different substrates in the turnover state

Mutation Substrate EPR parameter Ref.

Wild type Dinitrogen (N2 ) S = 1/2, g = 2.08, 1.99, 1.97 (107, 110)
α-70Val→Ala Propargyl alcohol S = 1.2, g = 2.12, 2.00, 1.99 (104, 105)
(HC≡CCH2 OH)
α-70Val→Ile Proton (H+ ) S = 1/2, g = 2.14, 2.00, 1.96 (103)
α-195His→Gln Methyldiazene S = 1.2, g = 2.08, 2.02, 1.99 (107, 108)
(CH3 N=NH)
α-70Val→Ala /α-195His→Gln Diazene (HN=NH) S = 1/2, g = 2.09, 2.01, 1.93 (109)
α-70Val→Ala /α-195His→Gln Hydrazine S = 1/2, g = 2.09, 2.01, 1.93 (106, 107)
(NH2 –NH2 )

These studies have advanced our understanding of where and how

substrates interact with the nitrogenase active site. Many other
questions remain to be resolved, such as
(1) Is the N–N bond broken in the trapped nitrogenous species
characterized so far?
(2) What is the level of reduction of the trapped states?
(3) What is the level of proton addition to the trapped states?
(4) Do intermediates migrate among the metals (Fe and Mo)
during the course of substrate reduction?
Answers to these and many other open questions will greatly
advance our understanding of this complex enzyme.

4. Conclusions
and Perspectives
Nitrogenase is a complex enzyme that plays a central role in the
global N cycle. Great strides have been achieved since its first dis-
covery to understand many facets of this complex system. These
advances are largely the result of the application of a wide range
of methods, many of which are described in this volume. Clearly,
much remains to be resolved about the mechanism of nitroge-
nase. Future advances will come from the application of the meth-
ods summarized in this volume, coupled with application of many
new methods. While the prospects for advancing understanding
of nitrogenase going forward are good, these advances are not
likely to come easily.
24 Yang, Danyal, and Seefeldt

Acknowledgments

The authors acknowledge the long collaboration with the Brian

Hoffman and Dennis Dean laboratories in advancing understand-
ing of nitrogenase. Work in the laboratory of the authors is sup-
ported by a generous grant from the National Institutes of Health
(GM59087).

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required for the simulation of kinetics of
Exploring the Variety of Random
Documents with Different Content
 YACHT REGISTER. 200 to 100 tons. Charmian 176 Myrtle
175 St. Kilda 175 Amalinda 174 BlueBeU 174 Ilona 172 Oriana 172
Tritonia 172 Christine 171 Fujiyama 171 Tarantula 171 Comet 170
John Fell 170 Angela 169 Frasquita 169 Medora 169 Stella 169 Vizir
169 Stanley 168 Torfrida 168 Anemone IV. ... 167 Lancer (The) 167
Kariad 166 Neva 166 Corinne 166 Jacinta 165 Groizeko-izarra ... 164
Dame Blanche ... 163 Lethe 168 Oimara 168 N^rine 162 Asterope
161 Dolores 161 Harlequin 161 200 to 100 tons. 200 to 100 tons. 8.
Ingeborg 8. Ondina ... Corisande s, Dorothy ... Dream s. Marcella... I
d. Barmore 8. Elfrida Jeannette Waterwitch 8. Fingal 8. White Eagle
8. Helen Feele Moonstone Brynhild Ada 8. Gal^jon Ivema (Ketch not
named) Fiona 8. Maretanza 8. Miriam 8. Filgrim Ro8e of Devon ... 8.
Belinda 8. Mandolin 8. Blandu8ia 8. Maruja 8. Team Aziola 8.
Cleopatra 8. EUinor 8. Sabine 161 161 160 { 160 160 I 160 I 169 ,
159 I 159 I 159 158 158 I 155 156 ! 153 I 162 ! 152 I 152 I 152 151
151 , 151 : 151 151 1 149 , 149 I 148 ' 148 I 148 I 147 147 147 I
147 I Oriole 8. Dragon 8. St. Hilda 8. Abafna Wendur 8. Dolores 8.
Margherita 8. Queen Margaret. . Sea Belle Alkelda Ylga 8. Bice
Tamesis 8. Zenobia Floral 8. Xanthus 8. Titania Galatea 8. Oberon
Heloise Zoraide Florinda 8. Lady Ghiga Phoebe 8. St. Frusquin
LavcDgro 8. Otter Gsea 8. Jennie Ranger Zala Astrsea s. Brabo 3.
Oaks 200 to 100 tons. 145 144 144 143 143 142 142 8. 142 8. 142 !
141 8. 141 140 140 140 139 139 138 137 137 136 136 135 135 135
135 134 134 133 133 133 133 132 132 8. 131 I Elfreda Lobelia
Arctic Cariad Silver Belle Suzon Wild Wave (The) Aventuri^re
Ayacanora Gitar.aII Jean Louis Lenore Seabird Alruna Dauntless Ina
Jullanar Madeline Scionda Abona Bona Chanticleer G«lert Ixia
Margita Ensa Gabrielle Huguenot Souvenance Thalatta Stulta Freda
Maria Amelia ... Dawn 130 130 129 129 129 129 128 127 127 127
127 127 127 126 126 126 126 126 124 123 123 122 122 122 122
121 121 121 121 121 121 ' 8. 120 120 119 200 to 100 toi LS. 119
Dora 8. Linda 119 8. Mehalah 119 Mirabel 119 8. Orelia 119 Rosita
119 8. Arrow 118 Iris 118 M'aza 118 lis Sunshine 8. Maja 117
Romany 117 St. Bernard 117 8. Tornado 117 8. Ulna 117 9. Lisette
116 8. Asthore 115 Four Brothers 9. (The) 115 8. Louise Alice 115
Zaneta 115 Zarita . 115 S. Eileen . 114 La Fl^che 114 . 114 H Queen
Mab 8. Tiercel 114 8. Mavis 113 S. Lia 112 Mallard 112 8. Powerful
112 8. Peregrine 111 Vida 111 Adolfo 110 Chloris 110 200 to 100
tons. .Leda 110 . Mary Louise 110 Molna 110 . Roxana 110 Volador
110 , Iliona 109 Isokol 109 PouiquoiPas? ... 109 Saint Alban 109
Bull-Frog 108 Florence 108 Planet Pilgrim... 108 . Samara 108 . Sea
Snake 108 Star of the Sea... 108 , Varina U^ Whisper 108 , Oreste
107 . Ceto 106 Kriemhilda 1'^ Reindeer 106 . Seahorse 10'» Aquila
105 Lola 105 . Nina H>5 , Eleanor 104 . Bna UH Hoyden 104 .Ren6
104 Sanspareil 104 Vol-au-Vent 104 Aileen 103 Galatea 103 Nereus
103 i^oo^le 1186 Digitized by
 )F YACHTS ARRANGED AOOORDING TO TONNAGE. 76U\J
JX} IKJKJ JAJUa. Auriga 102 Elena 102 Formosa 102 Hotspur 102
Laetitia 102 "Mi-rxr^na 1 MO lyjyj i«u Yo ifUnS. Nicandra 98 Ventura
98 s. Volga 97 8. WUd Flower 97 s. Abeona 96 Cicely 96 Erycina 96
8. Vista 95 Artemis 94 Samoena 94 Valkyrie 94 Cuckoo 93 8. Laurac
Bat 98 8. Alice 92 s. Amazon 92 Betty 92 Blue Rock 92 NelUe 92 8.
Seeschwalbe 92 Soft Wing 92 8. Tita 92 Bertha 91 Clutha 91
Dinsdale 91 Lais 91 8. Anemone 90 8. Annett.... 90 8. Chimaera 90
8. Eider 90 8. England 90 Galatea 90 Isola 90 8. La Dranse 90 8.
Linotte 90 100 to 75 tons. Loma ...90 s. Souvenir ... 90 Wanderer ...
90 Brunliilda ... 89 Isidora ... 89 La Vague ... 89 a. MachaAllah ... ...
89 8. Seabreeze ... 89 8. Sicilia ... 89 8. Skipjack ... 89 Heartsease ...
88 Irex ... 88 8. Julie ... 88 8. Romania ... 88 Bor^ ... 87 Cachalot ...
87 Flamingo ... 87 Kingfisher ... 87 Rosabelle ... 87 Florican ... 86
L'EtoUe ... 86 8. Primavera II. .. ... 86 Rhodesia ... 86 8. Silver Spray
.. ... m 8. Valkyrian ... 86 Veronica .... 86 8. Athene ... 85 Gladys ....
85 S.Lotus .... 85 Petronilla ... . .... 85 8. Thalatta ... 85 8. Leda ...
84 8. Wisy ... 84 8. Zinaida ... 84 100 to 75 tons. 100 to 75 tons.
Dream 83 Egret 88 B. Mandolin 83 Violet 88 Wave Queen 83 Wild
Wave 83 8. Assagai 82 Cheshire Cat 82 G-wendoline 82 8. La
Marguerite ... 82 Merganser 82 Star of the West... 82 8. White Friar
82 s. Adora 81 Columbine 81 Nixie 81 8. Primrose 81 Spindrift 81 8.
Adelaide 80 8. Alm^e 80 Fiona 80 Genesta 80 Gudrun 80 Maya 80
Onyx 80 8. Orient 80 Sula 80 Bird 79 8. Cristina 79 8. Devonia 79 8.
Hecla 79 Island Home 79 Maude 79 Meriden 79 Githona Resolute ,
AUona Bakaloum ..., B^niguet , 8. Danitza 8. Teshrifiyeh ... Aeolus
Dawn Druid Fenella 8. Gretchen Hesperia Kara 8. Nelly Reindeer s.
Waihi Yolande Flying Cloud 8. Gwendoline... 8. Johanna Kommodore
L'Esperance 8. La Perle 8. Valetta Vendetta 8. Whaup 1187 Digitized
by Google
The text on this page is estimated to be only 29.45%
accurate

76 to 60 tons. 1 8. Merel 70 Otterhound 70 8. 8. Saevuna

70 1 Vercing^torix 70 8.Aida 69 8. Carina 69 Catalina 69 8. 8.
Claymore 69 , Clorita 69 8. Etheldreda 69 8. j Gertrude 69 8.
KathUnda 69 Manica 69 8. 8. Paqueta 69 8. 8. Perlona 69 Satellite
69 8. Stradella 69 8. 8. Britannic II 68 Corsair 68 Bjeylane 68 8. 8.
Iri8 68 8. Marevo .-. 68 8. 8. Marita 68 Rubicon 68 8. Stellaura 68
Sundew 68 8. Susan 68 Windflower 68 8. Geraldine 67 8. lona . 67
8. 8. Princess Beatrice 67 8. 3. St. Quintin 67 8. 8. Stella 67 Varuna .
67 8. TAOHT REGISTER. 76 to 60 tons. ' 76 to 60 tons. , 76 to 60
tons. _u._ (Cutter not named) 67 . Adele 66 Ailsa 66 , Alba :'.... 66 .
Bonafide 66 I Brunette 66 , .Chanticleer 66 | Clunies-Ross 66 ,
Esperance 66 | . Hilda Muriel 66 i Minx 66 \ Serena 66 I . Avis 65 I .
Dora 65 ' Eldorado 65 | Flying Scud 65 , . Freya 65 | Lina 65
Mohawk 65 I . Samitza 65 Wyvern 65 . Adventuress 6-1 ; Celia 64 i
Chonita 64 Euphrone 64 I . Grand Duchesse I Marie Pavlovna 64 i .
Inyoni 64 . Ism^ ..: 64 I . Suzume 64 I .Tern 64 I .Thais 64 Banballl
63 . Belle 63 j Grand Duchess ... 63 I Margfuerite 63 s. 8. More Vane
63 | Pleione 63 8. Rita 63 8. Ruth 63 8. 8. Salamander 63!
SteMathilde 63 I 8. Zarnitsa 63 i Clytie 62 I 8. Highwayman 62 i
Roma 62 ' s Sheila 62 I Vanda 62 ' s. 8. Asteroid 61 8. Brioni 61 8.
Caroline II 61 Daphne 61 Gabrielle 61 8. Nomad 61 | Raven 61 s.
Viking 61 I 8. Wildfire 61 I Albatros 60 ' Corsair 60 | Gipsy Queen 60
i s. Harebell 60 ■ s. 8. Icicle 60 I s. Nevada 60 i s. Orion 60 8.
Paladin 60 | s, 8. Rover 60 ' s, Sakuntala 60 | s Tagide 60 | _ I 1188
Vittoria 60 Adriada 59 Aquila 59 Arethusa 59 ArziUa 59 Clanranaldll
59 Day Dream 59 Jessica 59 Laverock 59 Reverie 59 Susanne 59
Toto 51) Escargot 58 Goshawk 58 Las Palmas 58 Primrose 58 Thalia
58 Caprice 67 Charis 57 Cygnet 57 Druide 57 Gwynfa 57 Iris 57
Island Home 57 Jolie Brise 57 La Burgoine 57 Lady Olive 57 Magda
57 Marie 57 Smuggler 57 Surf 57 Angelina L 56 Banshee 56 Beluga
.....*. 56 8. DonRoderic 56 8. Finnuella 56 Luisa 56 Maris-Stella 56 8.
Maritana 56 Mosquito 0 56 Opal 56 8. TuUia 66 Undine 56 8. Auovri
(Dione) 55 Edwina 55 Elisabeth 55 8. Gftzelle 55 8. lona 55 Lygia 55
Mayflower 55 s. Nu-vot-eatschjo ... 55 8. Sibylle 55 Snowfleck 55 s.
Undine 55 British Queen 64 Creole 54 8. Firefly 54 Gaviota 51
Gwenyth 54 Irene 54 Irene 54 8. Manitou* 54 8. Osprey 64
Roseneath 54 Thea 54 8. Tou jours Prest ... 54 Vera 54 Warrior 64
LS. 8. Clanranald 63 Dolphin 53 Feronia 53 Hyacinth 53 8. lolair 53
Javelin 53 Stranger 68 8. Sylvia 63 White Slave 53 Avocet 52
Christabel 52 Clemenoe 52 8. Goeland 52 8. Sheila 52 Sirene 52
Stork ^« Thetis 8. Toro 8. Cysne Empress Fiery Cross ... Foxglove I
Frog ' 8. Kabyle ' 8. Kelpie 8. Lucille s. May Queen ... Norma
Peggotty Thele Velsa Vesta (Taoht buildi]
 LIST OF YACHTS ARRANGED ACCORDING TO TONNAGE.
60 to 30 tons. Ada 50 Armorel 50 Brenda 50 . Hansa 50 Marinetta 50
Merlin 50 Miriam 50 Moonrise ^50 Peri 50 St. Louis 50 Shearwater
50 Sphinx 50 Sulaire 50 Thora 50 Twilight 50 White Rose 50
Charmer 49' Dortha 49 Girofl^ 49 Ingeborg 49 Leander 49 Panliette
49 Palmoea 49 Pleiad 49 Snapshot 49 Solrac 49 Tennessee 49 rhekla
49 Zareba 49 Albatross 48 Brada 48 :>irina 48 Marietta 48 3hotah
48 50 to 30 tons. 8. Daphne 48 s. Hans 48 8. Ivy 48 s. Orell 48
Palatina 48 Sea Bird 48 s. Senta 48 s. Siren 48 8. Vacuna 48
Wanderer 48 Waveney 48 Wild Flower 48 Yeldiz 48 Ailsa 47 8.
Bantam 47 8. Carola 47 8. Electric 47 Etona 47 Horla 47 lerne 47
Lncida 47 Maria 47 Milenka .-. 47 s. Onde 47 s. Scalpa 47 8.
Semiramis 47 Wenonah 47 Woge 47 s. Wyvem 47 Anaconda 46 8.
Berceuse 4fi Coquette 46 Dolphyn 46 Echo 46 60 to 30 tons. 50 to
30 tons. Fawn 46 Heron 46 Magdalen 46 Othona 46 Vindex 46
Wulfruna 46 (Sew. Sch. not named) 46 Aglaia * 45 8. A^nle 45
Ayame 45 8. Bandit 45 Challenge 45 Costanza 45 Diad^me 45 Dione
45 Fortuna 45 Free Lance 45 G-odwit 4n Hypatia 45 Imatra 45 s. Ino
45 8. Irene 46 Lena 45 Neotia 45 Nereid 45 s. Scud 45 I Sibyl 45 I
Sirena 45 i s. Snowflake 45 I Watergeus 45 I 8. Wildfire 45 | ! WiUe
45 I I Tamato Damashi 45 (Cutter not named) 45 8. Australia 44
Bedouin 44 Clitunno 44 Clutha 44 8. Diana 44 8. Greyhound 44 Hera
44 LiUth 44 8. Maia 44 8. Mandolin 44 Marcelle 44 Mavis 44
Potamide 44 Prima Donna 4i Roebuck 44 Bona 44 Rustler 44 Sea
Queen 44 8. Sparkle 44 Vanadis 44 Wayfarer 44 Willow Wren 44
Windward 44 Xenia 44 Alibi II 4.3 Anna 43 Annasona 43 Astarte 43
8. Bessie 43 Bronwen 43 Canopus 43 Elbe 43 Est^rel 43 50 to 30
tons. 1189 . Milranda Mystery Nara . Pens6e . Peridot Ren6e-Marie
Reverie . Sloughi White Heather ... . Alcyone Alice . Antidote Calisaya
. Cormorant Ellen Era . Firefly Gazelle . Irene . Larkan Latona
Moonbeam . Saxon Terpsichore . Valda . Venture Vigoma Wanderer
Zenobia Dragon Elsa Fox Ingeborg Koh-i-noor f r^r^r\lr> Digitized
by VrjOOv IC
 YACHT REGISTER. 50 to 30 tons. Lady Blanche 40 Mariana
40 Nadeschda 40 Nellie 40 Norman 40 Oasis 40 B. Paghan 40 8.
Parole 40 Phryne 40 8. Stephanie 40 Stephanotis 40 Vigilat 40 8.
Yvonne 40 (Cutter not named) 40 (Sew. Sch. not named) 40 Aileen
39 Alceste 39 8. Alexandra 39 Anaconda 39 Ariadne 39 Atalanta :.
39 Bear 39 8. Bulrush 39 8. Chim^re 39 Courlis 39 Dinorah 39 8.
Eulima 39 Fleur-de-Lys 39 Geisha 39 Gk)8samer 39 Ilona 39 Lnciole
III 39 Luisa 39 Miranda 39 Neaira 39 Nevermind 39 Penitent 39
Saint Her v6 39 Trumpf 39 s. Turbinia 39 Zampa 39 8. Bohemian 1
38 8. Bronzewing 38 Cabarfei 38 8. Curlew 38 8. Dorothy 38 8. Fifi
38 8. Giooonda 38 s. Harbinger 38 Inyoni 38 8. Lady Nell 38 Lally 38
s. Lily 38 8. Miranda 38 Nixe 38 Oscar & Virginie 38 8. St. Hubert 38
8. Star 38 Amaryllis 37 Caprice 37 Coralie 37 8. Dormouse 37 I 8.
Douira 37 I Echo 37 I Gauntlet 37 I Giroila 37 Griselda 37 Induna 37
Luna 37 Marguerite 37 Mersey 37 8. Nama 37 8. Reine Margot 37 8.
Ruby 37 Saint Ren^ 37 S.Sol 37 Storm 37 Sunbeam 37 s. Sunmaid
37 Sunset 37 Svanhild 37 Sylphide 37 Thca 37 8. Veslemoy 37 Viera
37 Walwin 37 8. Welcome 37 Woodbine 37 Alvor 36 Aspasia 36
Avellll 36 Brilliant 36 s. Chrysoprase 36 Dauntless 36 Dawn 36 8.
Eaglet 36 Gloria 36 8. Griffin 36 Griso 36 8. Harrakka 36 8. Insua 36
8. Ladybird Lesbia Maid of the Mist Maladetta 8. Marinette Moorak
Najall Nebula 8. Nirvana 8. Primrose Quihai 8. Bona Tambourine
Viking Waterwitch s. Ytene s. Arethusa Astrid Bianca Cachalot
Chevreuil Claudian Dandelion Deirdr6 Dianthus Edelweiss Elektra!..;
S.Ella 8. Evangeline Fawn Forsa Fox Hound Glance Glimpse 50 to 30
tons. Grayling 35 8. Hirondelle 35 lanthe 35 8. Janet 35 Lucina 35
Marolga 35 May Queen 35 Merrie Duchess ... 35 Merrythought ... 35
Morning Star 35 Nautilus 35 8. Northumbria 35 Senga 35 Sorceress
35 Stella 35 8. Thistle 35 Wave 35 Wave Queen 35 Yann 35 8.
Zenobia 35 Aideen 34 Albatross 34 Althea 34 Banba 34 8. Berthic 34
8. Chimera 34 S.Clara 34 Clymene 34 Comus 34 8. Excelsior 34 Gala
34 Humming Bird ... 34 Lacerta 34 Lyonnesse ......... 34 Digitized by
Vrr 50 to 30 tons. s. May Queen 34 MenHir 34 Merrymaid 34
Meschaceb^ 34 8. Nadeschda 34 OUve 34 Prima Donna 34 s.
Scarab^ Si 8. Sl^eandhu 34 8. Vallota S4 8. Vesta 34 Alruda 33 Bella
Donna SS Cyrano 33 Dora 53 Ellida r. SS Flame SS 8. Gael $3
Galatea SS Gipsy SS 8. Gladys 33 Helen SS Marguerite 3S Marigold
5S Mayfly SS 8. Moira 88 Namenlos SS Neptune SS 8. Phoenix 88
Sea Mew 83 s. Sea Nymph 88 Selene 88 Sommerfuglen ... SS 8.
Siinl^eam SS e
 LIST OF YACHTS ARRANGED ACCORDING TO TONNAGE.
60 to 30 tons. Surpreza 33 8. Suzon 33 8. Una 33 8. Ver&no 33
B.Vesta 33 8. Violet 33 Volunteer 33 Zephyr 33 B. Airam 32 Anna 32
Asphodel 32 s. Balaena 32 a. Britannia 32 Brunette 32 Garissima 32.
I Colapmi 32 Coral 32 i Daisie 32 | B. Excelsior 32 I Gipsy 32 | 8.
Glow- worm 32 , Hilda 32 I Licht Teyreden ... 32 | 8. Luath 32 |
Marie Louise 32 i 8. Mars 32 { Nan 32 | Nemo 32 i 8. Princess
Beatrice 32 I Puffin 32 I Rose 32 I Rosemary 32 I Rubi 32 St.
Georpres 32 | 50 to 30 buiio. Senta 32 Sylvia 32 8. Una 32 Widar 32
Airy-Mouse 31 Andromeda 31 Argo 31 Ariadne 31 8. Berceuse 31
Bettina 31 Candor 31 Capri 31 B. Caroline 31 Gymbeline 31 s.
Dandie Dinmont ..31 Dark Secret 31 s. Dorothea 31 Dunearl 31
Eclair 31 Excelsior 31 8. Fashion 31 s. Fireflash 31 Gitana 31 Kiama
31 Laurea 31 May Mom 31 Meg Merrilees 31 Nautilus 31 B. Nykr 31
8. Osprey «... 31 8. Scaup 31 Sirius 31 8. Starlight 31 Thyrza 31 UU
bU OU UUULO. Kf\J U\J 4Si\J lA^UO. Trilby .. 8. Vittoria 30 to 20
tons. Aggie Avon s. Bluebell ... 8. Britannia Calumet ... Carib Cetacea
I Chevalier... I Curieuse ... ' 8. Dauphin ... j Dolfyn I Doris I Embla I
Geisha I Gerda I Guillemot I 8. Harry I 8. Hermione I 8. Ingomar ...
Isis 8. Isola Bella 8. Ixolite Japonica ... 31 I Joyeuse 30 31 I KUmeny
30 s. Lei 30 Liebe 30 Lust 30 Marigold 30 Mathilda 30 s. Maud 30 s.
Meteorlight 30 Mildred 30 Nesta 30 Osiris 30 Petrellen 30 S.Philomel
30 Polly 30 8. Rambler 30 s. Rapide 30 s. Sequana 30 s. Solskin 30
Trilby 30 8. Undine 30 s. Volunda 30 Walkyrie 30 s. Adonis 29
Armorel 29 s. Atalanta 29 s. Chinook 29 Chiquita 29 Crusader 2^
Dauntless 2 8. Diane 2 Donnetta 2' 8. Emma 2! Enigma 2 30 30 30
30 80 HO 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 SO 8.
Frances 29 Hesperus 29 Hirondelle 29 Hoopoe 29 Iduna 29
Kommodore 29 Lady Harvey 29 LaBsie 29 Leonid 29 s. Liline 29 s.
Maid of Athens ... 29 Nan 29 Nynee 29 (Enanthe 29 s. Om^ga 29
Opah 29 Omen 29 Ortrud 29 s. Puffin 29 Rendezvous 29 Reverie 29
Rhoda 29 St. Helen 29 8. Silver Star 29 Spring 29 Stella 29 Stella
Maris 29 Sybil 29 a TttiH-Qi. 9Q 8. Wy vern I Abeona .. 29 28 28 28
28 Avel Bato s. Cerqueira Lima .. Champion 28 B. Chinook 28 Courier
28 Cyclamen 28 De Wulp 28 Drift 28 8. Duenna 28 Eider 28 s? Ester
28 Etendard 28 Farspach 28 Fyona 28 8. Grebe 28 lanthe 28 Ilwo 28
s. Iris 28 Irma 28 Jacqueline 28 Magic 28 8. Marjorie 28 Mayiy 28 s.
Meeuw 28 8. Meeuw 28 a UTATia 9fi Digitized by 1191 Google
 TAOHT REGISTER. 30 to 20 tons. Rover 28 8. Sachem 28
Speedwell 28 Svint 28 Topsy 28 Twilight 28 Ville de Marseille 28 8.
Vivara 28 8. Waterwitch 28 8. Whimbrelll 28 WUdDuck 28 Xanthe 28
Zoraida 28 AUegra 27 Mma 27 Alpha Beta 27 Anemone 27 8.
Arcturu8 27 Armide 27 Avonia 27 Beduin 27 8. Bice 27 Blenda 27 8.
Britannia 27 8. Cabarfeidh 27 8. Chipmunk 27 Chough 27 8.
Cigarette 27 Cypris 27 Eileen 27 Erato 27 8. Express 27 8. Firefly 27
Foam 27 30 to 20 tons. 30 to 20 tons. 8. Forel 27 ] 8. Gazelle 27
Gladys 27 ' Glance 27 | Gracie 27 8. Hermione 27 | Kaemka 27 i
Kittie 27 I Klabautermann ... 27 Klomp 27 Lenore 27 8. Lurline 27
Mabella 27 Marina 27 S.Medina 27 Medj6 27 8. Milhafre 27 Milianne
27 Mimosa II 27 Molly 27 8. Nellie 27 Nora 27 Givana 27 Othona 27
Pauline 27 Peregrine 27 Persephone 27 Silver Crescent ... 27 Siola 27
Sone 27 8. Tarpon 27 8. Tartina 27 Venture 27 Viv 27 Vivandiere 27
Wanderer 27 White Seal 27 Zvezda 27 Archina 26 8. C6celle 26 8.
Cruban .- 26 Crusader 26 8. Cypris 26 Dinah 26 8. Donna Lucia 26
Dorothy 26 Hertha 26 8. Ithuriel 26 Juno 26 Kelpie 26 8. Kettledrum
26 Lhasa 26 8. Lily 26 Lyn 26 8. Malvina 26 May Queen 26 S.Miriam
26 Moraima 26 Nell 26 8. Nordisa.. 26 Otter 26 Phoenix 26 Bosetta
26 Scart 26 Seadrift 26 Seaweed 26 Siesta 26 Spray 26 30 to 20
tons. Storm 26 Tzigane 26 8. Vidette 26 8. Volage 26 Wyvern 26
Yalch'ik III 26 8. Mglh 25 Anemone 25 Arrow 25 8. Busy Bee 25
Casita 25 Charm 25 8. Comet 25 Coventina ......... 25 8. Coyote 25
Daisy 25 8. Dart 25 Diana 25 Dorindy 25 Dryaden 25 8. Edina 25
Electra 25 8. Elsie 25 8. Flamboyante 25 8. Gertrude 25 Hela 25
Isolde 25 Keenaghmore 25 8. Lerici 25 Lolah 25 Maritana 25 May 25
Mignon III 25 Moonbeam 25 30 to 20 tons. Muguet 25 Nanna 25
Niari 25 Nita (The) 25 8. Rambler 25 Rigel 25 8. Roseberry 25 Saga
25 Starling 25 Storm 25 Sylphide 25 8. Telefon 25 Triton 25 Undine
25 Vega 25 Velzie 25 Vera 25 8. Violet 25 Volhouder 25 Waratah 25
8. ZutIV 25 Achilla 24 s. Ada 24 ^olia 24 Albatross 24 Almida 24 6.
Ashburn 24 Bostonia 24 8. Bulldog 24 Canada 24 Carlota 24' 8.
Caversham 24 Clementine 24 Cyclamen 24 30 to 20 tons. 8. Cygnet
24 ! Daring 24 Golden Hind 24 Idalia 24 Jessie 24 Leisure Hour 24'
Leemona III 24 ■ Maria 24 s. MeUta 24 8. Metohta 24 . 8. Miramar
24 t Mirza 24 Nada 248. Nymph 24 | Otter 24 Sea Bird 24 8.
Serpolette 24 Spider 2i Sturmvogel 24 Sweetheart 2* Tammie Nonie
... 24 Tolosa 24 Verbena 24' 8. Volage 24 Waitangi 24 8. Wayward
24 Winifred U 8. Yvette 24 Zaria 24 Zayda 24 Zephyr 24. Zora Ul
Zwerver 24'; Almom 21 :■ 1 v^oogie 1192 Digitized by
The text on this page is estimated to be only 22.95%
accurate

LIST OF YACHTS ARRANGED AOCORDINO TO TONNAOR,

30 to 20 tons. Belami 23 Bingo 23 8. Boh^mi^n 23 Bride 23 Carida
23 Caroline 23 Cerigo 23 Colleen 23 Embla 23 a. Etoile 23 Falken 23
Florinda 23 Giovanni 23 Gne-Lou-Zy 23 8. Halcyone 23 Heron (The)
23 8. lernia 23 Inyala 23 Jannette 23 Jess 23 Job-Jeanne 23 s.
Kingfisher 23 Krydseren 23 Loiterer 23 May 23 May 23 a. May Queen
23 Mistral 23 Najade 23 Nirrana 23 No. 409 23 Ondine 23 a Platypus
23 Beguia 23 30 to 20 tons. Rover 28 Roxane 23 Sans Souci 23
Sapphire 23 Sayonara 23 Scout 28 Silver Foam 23 Swan 28 Titania
28 Uteha 28 Vigilant 28 Volatile 28 8. Volpicelli 23 Vreda 23 8. Wapiti
28 8. Ythan 23 Zeuthen 28 ZigZag 23 8. Alma 22 8. Amoena 22 8.
Asmod^ 22 0. Boomerang 22 Boulinet 22 Caprice 22 Chita 22 Circe
22 i I Cotillion 22 i I 8. Daisy 22 I ' Dorothy 22 Eagle 22 ; Elek 22 1 .
8. Ellen 22 Elsie 22 Estrella 22 30 to 20 tons. 8. Etinoelle Eva
Faerdor 8. Fairy Queon . Fingal 8. Fleur de Franc Flying Cloud . 8.
Gipsy 8. Gleaner a. Gondolier .... Helen If/)15ne Irmgard Ivy 8. Julia
8. KittitwfUco .... 8. Lili Mars Mattie Mimine Mimosa Mina Moriana
MyoHOtis s. Nemo Nenuphar II. . Ninon Ober Spree .... Olga
Patience s. Pleion« s. Psycheia , s. Puffer Crhii) .. Revenge , 30 to 20
tona. 30 to 20 tonii, no to »0 tonH, Digitized by Google
 YACHT BEGISTBR. Alcina 20 Avalon 20 Bacchus 20 8. Bean
20 Bee 20 i 8. Blunderbuss 20 | Butterfly 20 Carmen 20 i 8. Casilda
20 \ 8. Colombine 20 Coralie 20 | Cormoran 20 Druidess 20 | S.Ella
20 I s. Elysia 20 ; Ermelinda 20 Etona 20 Falke 20 Fin-de-Si^cle 20
8. Flaminpfo 20 8. Formosa 20 8. Frolic 20 Frolic 20 Gadfly 20
Gardenia 20 Getingen 20 Grig 20 Halcyon 20 Heartsease 20 Helena
20 Ilex 20 Irene 20 Jris 20 Jessamine 20 8. LaCrevette 20 8. La
Guepe 20 Laurita 20 Little Em'ly 20 Lizzy 20 Mabel 20 Madcap 20
Maggie 20 Maggie 20 Magnolia 20 Maia 20 Maritana 20 Medusa 20
8. Minnehaha 20 Miss Mary 20 Moira 20 Molly 20 Mona 20 Nautilus
20 Narval 20 8. Nina 20 s. Oasis 20 (Enone 20 8. Olivette 20 Pagan
20 Penelope 20 Playmate 20 8. Powan 20 Ptarmigan 20 Pukaki 20
Rob Roy 20 Rover 20 a^-m,^-^^-,u% on ) to 15 tons. Scorpion 20
Shutschok 20 Sirius 20 Stella 20 Stella 20 Sweet Nell 20 Tanit 20
Thyra 20 Tina 20 Uarda 20 Undine 20 Undine 20 Vanessa 20 Vanity
20 Varani 20 s. Vera 20 Verve 20 Wanderer 20 Watergeus 20
Watersprite 20 Wayward 20 s. Wilhelmina 20 WiU o' the Wisp... 20
Windward 20 Wyvern 20 Xela 20 Yvalda 20 Zargo 20 Acasta 19
Albatross 19 Alfreda 19 Arendal 19 Ariel 19 Astrid Augusta Auguste
Elizabeth Avoset Banba Black Rose B'nita Boby Buccaneer s.
Buccaneer Buzzfly Caltha Cayman Charm Chiquita Chiripa Day Dawn
Domikll Eileen Ella Ellen EUora Elodie Elspeth Emily Evadne
Fliegende Wolke... Fri Friso 8. Infemet Kvik 8. La Lys Linalll 19 19 19
19 19 19 19 19 19 19 19 I 19 19 19 19 19 19 19 19 19 19 19 19 19
s. 19 I 8. 19 19 19 19 19 19 19 19 I in I Maga 19 Magnolia 19 Maria
19 Marmoset 19 Masonic 19 Maub^he 19 Metis 19 Miczi 19 Minnie
19 Mireille 19 Molita 19 Mona 19 Musidora II 19 Nancy 19 Nautilus II
19 Norah 19 Orion 19 Oriva 19 Pansy 19 Pollie 19 Poppy 19 Rex 19
Rover 19 Runnach 19 St. Georges 19 Salammbo 19 Scamp 19
Seaweed 19 Shark 19 Sidora 19 Solace II 19 Sooloo 19 Spray 19
Digitized by (^oogle
The text on this page is estimated to be only 28.29%
accurate

LIST OP YACHTS ARRANGED ACCORDING TO TONNAGE.

20 to 15 tons. s. Falcon 18 ] Flirt 18 I s. Forest Fly 18 ' FnMa 18
Georg^e. 18 \ b, B Gladys 18 i QUde 18 , s. Gull 18 a. Hammonia 18
| g, Hebe 18 s. H^ldne 18 Hinda 18 Inca 18 Kajol 18 Kate 18 Kemick
18 Kite 18 LallaRookh 18 ' L'Esperanoe 18 t Lily (The) 18 , Little
Windflower 18 Loreley 18 i Louie 18 ' Lnroime 18 | Marga 18 I
Margery Daw 18 ' Matangi 18 ! 8. Meteor 18 Mollihawk 18 i Moretta
18 B. Nancgr 18 Narcissus 18 8. Narwhal 18 Nathalie 18 20 to 15
tons. Nautilus Nona Oberon , Petula Princess Rita Rob Roy Rowena
Roy Scout , Seal Sealark Singora , Solveig , Spero Sundog
Sweetheart ... Syrene , Tarasque , Trio Tumleren , Turk , Valerie ,
Vedette Viking Violet Vixen Whimbrel .., White Stork White Wings
Xarifa Yum Yum ... Yvonne , Zanzara 18 18 18 18 18 18 18 18 18 18
18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 ! 18 j 18 1
18 I 18 I 18 I 20 to 15 tons. s. Zelda Zephyrine., Adda Andree
Apertyx Arlequin Aziad^ Bella s. Bogie s. Brisei's Camargo Canada
Chough Content Dolly Varden .. Dove Dryad Eagle Elly Eperlet
Etincelle Eva Mary Fan-Tan Faun Florence Foruak Fre^a s. Galinette
Garm Genesta Guenora Harriette Ann Hygeia 17 17 17 17 17 17 I I
18 I 18 j 17 I 8. 17 , 17 Ji . 17 s. 17 ! 17 17 17 17 17 17 17 17 17
17 17 17 17 I 8. 17 I 17 17 ' 17 ' 17 ; 17 ( 17 20 to 15 tons. 20 to
15 tons. I I Ibis 17 Ida 17 ' Imogen 17 | Inyala 17 i lola 17 I Island
Queen 17 ' Kiwi 17 1 Lamorna 17 Lassie 17 I Lurette 17 I Mimosa 17
j Minnie 17 Myrtle 17 Nanna 17 Norman 17 Olga 17 Olivana 17 I
Ondine 17 j Penguin 17 | Phantom 17 Puffin 17 ; Rarahu 17 ! St.
George 17 Sans Souci 17 ' Shamrock 17 Sinclair 17 Sonata 17 |
Sooloo 17 I Sylph 17 I Teresa 17 ' Thisbe 17 | Tirolia 17 | Turquoise
17 i Ulala 17 ; I Una Viking Vivia Volunteer Wasp Whisper White
Heather ... a. Winona g. Woodcock Acnaib Alert s. Alipha Anita
Asthore Athene B. Bianca 8. Cairngorm 8. Calumet 8. Caprice Celt
Clementine Clodagh Coila Coot Corinne Crisis CristoforoColombo s.
Cuckoo Curtsy Daffodil Dalila D. F. C Diamond Dis Digitized by 20 to
15 tons. 17 i. Djeiyrely 16 17 Drift 16 17 I .s. Dunlin 16 17 1 Eagle
16 17 ! Eclipse 16 17 I Eileen III 16 17 1 Elspeth 16 17 Endrick 16
17 I Enid 16 16 Ensay 16 16 Eureka 16 16 Fiona 16 16 Firefly 16 16
Fleur-de-Mai 16 16 Florence 16 16 s. Foam 16 16 8. Frou-Frou 16 16
8. Galatea 16 16 Glendoveer 16 16 ' Griselidis 16 16 , Gurly 16 16 I
Halcyon 16 16 ' 8. Hawk 16 16 8. Helix 16 16 Henny 16 16 lona 16
16 Iota 16 16 ' s. Irene 16 16 Iris 16 16 , ». Ittekc 16 16 1 S.Jane
.'... 16 16 Kingfisher 16 16 Kismet 16 Coquette I ^6t' lIQft
 YACHT REGISTER. 20 to 15 tons. 20 to 15 tons. 15 to 10
tons. Lady Alioe 16 lianneret 16 Lincoln 16 8. Lodona 16 Luna 16
Maagen 16 8. Madcap 16 Maggie 16 Magnolia 16 Maple Leaf 16
Maraquita 16 I Margarita 16 Marie 16 Marie H^ldne 16 8. Marie
Louise 16 Maritana 16 Masaniello 16 Maude 16 Melita 16 8. Mermaid
16 MouBm^ 16 8. Mythra 16 9. Nadine 16 Nanette 16 1 8. Nautilus
16 i Nina 16 Nyama 16 Ormonde 16 Otter 16 Redgauntlet 16 Red
Lancer 16 Redwing 16 Rivale 16 Sarcelle 16 Sassenach SauDterer 8.
Seal 8. Serapis Silver Spray 8. Sonachan 8. Speedwell 8. Speedwell
8. Sphinx Titti 8. Undine Valborg Valerie Valkyrien 8. Virginia 8.
Volage Volante Wave 8. White Rose of York (The) Wild Duck
Wilhelmina Woinora Zelma 8. Zouave I 16 16 16 16 ! 16 I 16 , 16 16
I 16 ' 16 ' 16 16 I 16 16 '■■ 16 16 I 16 16 16 16 16 16 16 16 15 to
10 tons. Aborigine 15 6. Ada 15 Alca 15 Alyette 16 Amulet IT 15
Ariadne 15 Barabel 15 Beatrice 15 Bel-Ami 15 Betchen 15 Bonita 15
Breiz 15 8. Brunette 15 , Bullfinch 15 Cerise 15 Chicken 15 I Contest
15 I Countess 15 , Cybele 15 Daisy 15 i Donna Sylvia 15 ' Duen 15 a.
Eclair lo I Eclipse 15 ; Eileen IV 16 i EUide 15 Endymion 15 Etincelle
15 i s. Falcon 16 ; Firebrand 15 [ Flora 15 ; Florence 15 Frou Frou 15
j Gem 15 ] I Grane 15 Granta 15 s. Gustaf Adolf 15 Heather Bell 15
Hebe 15 Hepsie 15 Hermes 15 8. Hopbloom 15 8. Hoyden 15 lanthe
15 Ida 15 Ida 15 Ignota 15 Ildeg^nda 15 Hex 15 Irene 15 Irenell 15
Jeannette 15 Jetta 15 Joan 15 Joanna 15 Karen 15 Kite 16
Kommodore 15 s. La Rondona 15 Lady Winifred ... 15 Leif 15
L'Esperance 15 8. L^vrier 15 8. Libellule 15 Lilith 15 Luna 16 Maagen
16 Mabel 15 15 to 10 tons. Mariatta Marionette .... Marsouin Martha
May Mermaid Minnehaha .... Monara 8. Mon Jean .... Morgana B.
Mouette Nell Neva 8. Novice 8. Ondine Parkeler Patronita Petrellen 8.
Pioneer Plover 8. Queenie Rainbow 8. Remember Rosemary Rosetta
Rosie Rota B. Saint Maurice.. Saint Tudy 8. Saladin , 8. Sarcelle 8.
Soylla Shamrock ..... Sirdne 15 15 15 15 15 16 15 15 15 16 16 15 15
16 16 15 16 16 16 15 15 I 15 16 15 16 1.5 15 15 ; 15 15 15 to 10
tons. Songstress Sperwer Spindrift Sport Storegut Sunbeam
Sunshine . Sword Fish Thalia Theta Tullia Tfimmler III.... Urania
Vega Venus Victoria . Violet Virocca Wayward Tanniok . Zingara Ziu
(.Sew. Cutter not ] named) Alumina . Alumlnia Amelia Amy . Angelo
Ariadne Arminio Ava 1196 uigitizea oy t3^0r
The text on this page is estimated to be only 23.52%
accurate

LIST OP YACHTS ARBANGKD AC5CORDIN6 TO TONNAGE. I

16 to 10 tons. 16 to 10 tons. 16 to 10 tons. BagateUe 14 I Banshee
14 ' s. Breeze 14 I 8. Breezie 14 | Bnocaneer 14 Caiman (Le) 14 | s.
Carina 14 I Chndo 14 s. 8. Cintra 14 | Cisne 14 i Clare 14 i e. Colleen
Bawn 14 i B. Coquette 14 | Corona 14 Curlew 14 De Wildeman 14
Debonair 14 Dione 14 Dorothy 14 Elaine 14 8. Electric 14 Elfin 14
Elgriva 14 Blphy 14 Bob 14 Bros 14 Eudora 14 i Fairlie v 14 Fairy 14
Faith 14 Falken 14 Qeraldine U . GipHy Queen 14 Girleeu 14 Glad 14
Gloriana 14 Greta 14 , Ganlog 14 Helene 14 . 8. Innisfallen 14 Irene
14 b. IriB 14 Juanita 14 > Juliette 14 , Kathleen 14 , Kathleen 14
Kelpie 14 ! King^eher 14 | b. KirBten 14 | Kosmos 14 { La Dives 14
,i s. Lady G«orgiana ... 14 1 Leila 14 ^ Lizzy 14 < Lorna 14 | Loma
Doone 14 i Lucie 14 ' Maiblume 14 , Maid Marion 14 i Marguerite 14
; Mathilde 14 May 14 Mayflower 14 Minkie 14 Mobile 14 Moonflower
14 Nadejda 14 Namouna 14 , Neta 14 Niobe 14 Oithona 14 i Panther
14 Paroquet 14 Pastime 14 j Peri 14 Perkun 14 Petrel 14 i Primrose
14 PrizrAkll 14 Rita 14 , Rita 14 ' Rona 14 , Roonette 14 j Rover 14 I
Roxane 14 i Rui^iue 14 ' Scoter 14 i Semoulette 14 < Senta 14 i
Sentinel 14 Sigrid 14 ; Squaw 14 ! Stellaura 14 ! Storm Queen 14 I
Sylphe 14 i Sylvia 14 Thyra 14 Tocayo 14 Valentine 14 ! Veszmadar
14 V^vette 14 Victor 14 16 to 10 tons. Wahbewawa 14 Walktbre 14
8. Waterwagtail 14 8. White Heather ... 14 Woodcock 14 Wych 14
Yucca 14 Yvonne 14 Achill 13 Agnes 18 Aiguillon 18 , 8. Alerte 13 i
Alexa 13 i Alfhild 13 ! 8. Alice 13 I Alice IS I Amoretta IS ! Aorere IS
Ariadne IS 8. Arion IS 8. AsBynt IS < Attila IS I Baldur 13 ; Blonde
IS , 8. Bumble Bee IS i 8. Bus (The) 18 ; Buvar 13 , Chang and Eng
... IS | Charm 18 i Clara 13 ' Cruiser 13 8. Cygnet 13 Cynthia 13 Dina
13 16 to 10 tons. Dione 18 ! Doris 13 i Dorothy 13 i Ebor 13 s. Elfin
13 8. Elisa 18 Encore 13 Ethel 13 I Fama 13 , 8. Faniska 13 Fantom
13 Farewell 13 \ Fata Morgana 18 1 Fee 18 I Frigg 18 Gipsy 13 I
Gossip 18 ; 8. Growley 18 i Gulli 18 I Heron 18 Hevella 18 Hilda 13
Hirondelle 18 Hyla 18 Ibis 13 Ida 13 ! Ina 13 8. Irene 18 j Iris 13 I
Isa 13 Isita 13 ' Juno 13 Kate 13 Kathleen 18 I 16 to 10 tons. Kismet
18 B. Kismet 18 Kittiwake 18 LaCigale 18 , Little Nell 13 Lola 18 I
Lucifer 13 Mabel 13 s. Mabelle 18 B. Malariki 18 Marianne 18 Mariv)
II 13 8. Mayflower 13 May Marie 13 Minerva II 13 Minota 13 Muritai
13 Mynah 18 8. Myra 13 Naiad 18 Nautilus 13 Nautilus 13 Nedda 13
Nereus 13 Nike II 18 Nimble 18 Ninette 13 Njala 18 8. Norma 13
Oberhavel 13 Onward 13 Orchid 13 Osprey 13 Osprey ""* iia?
 YACHT REGISTER. 16 to 10 tons. Pattie 13 Pax 18 Petrel 18
Piccaniny 18 Picua IS PingoiiJn 13 8. Pirouette 13 Puritan 13
EedWiug 18 Repose 13 Ricochet 13 Romania 13 Romany Lass 13
Sapphire 13 Sappho.... 13 Sheldrake 13 Sirdar 18 Snowbird 13 fi.
Snowdrop 13 Starkodder 13 Stella 13 Suzette 13 Svanhild 13 Tess 13
Thistle 13 Trirumpf 13 Union 13 Vera 18 s. Verada 13 Viking 13
Volant 13 Volta 13 Westra 13 '^e 13 16 to 10 tons. Winfreda 13
Woodcock 13 TumYum 13 (Ywl. not named) 13 Agnes 12 Alektor 12
Arrow 12 Atalanta 12 Atila 12 Avenell 12 Ayala 12 8. Beryl 12 8.
Betty 12 S.Birkenhead 12 8. Bobs , 12 Brena 12 Bruna 12 8.
Catherine H 12 s. Cecile 12 Ghana 12 8. Ch^re Amie 12 Oitara 12
Olymene 12 8. Comus 12 Cruiser 12 Curlew 12 Cynthia 12 Cypraea
12 Dakotah 12 Dione 12 Dobhran 12 Dolphin 12 Dream 12 16 to 10
tons. Duschka 12 Edie 12 Edith 12 8. Elf 12 8. Elfin 12 Blin 12 Elsa
12 Eos 12 Eurynome 12 Eva 12 8. 1'ilibuster 12 Florence 12 Flyaway
12 Forella 12 Freya 12 Fri 12 Fulmar 12 8. Gallia 12 Gannet 12
Gascon (Le) 12 Gefion 12 Germaine... 12 Gluckhauf 12 8. Gwynedd
12 8. Haidah 12 Hanseat 12 Hedwig 12 Helen 12 Henriette 12 8.
lanira 12 Ilka 12 8. Invicta 12 Irene 12 Iris 12 16 to 10 tons. Isabelle
12 Isard 12 Ito 12 8. Jap 12 Jhilum 12 Kampioen 12 Kate 12 Kibitz
12 Kilen 12 8. Kerrigan 12 Lalage 12 L'AUegra 12 La Mouette 12
Leda 12 Leonore 12 Libelle 12 Lina 12 Lufra 12 Luzette 12 Maida 12
Maleine 12 Malista 12 Maraquita 12 Marguerite 12 Marina 12 8.
Mayflower 12 8. Merlin 12 Minniehaha 12 8. Monaco 12 Myrtle 12
Naima 12 Nella 12 Nerissa 12 Nerissa 12 8. Net Ne^ Nil Njc Noi Olg
Oh On Ozc Par Pai Per Pet 8. Pin Pla; Pol; 8. Prii Psy Ret Rul Sai( San
San Seal Scot Seal Seci Ser] 8. Stai Stel Tan Tarj 8. Ten The 1198
Digitized by Google
The text on this page is estimated to be only 29.43%
accurate

LIST OP TAOHTS ARRANGED ACCORDING TO TONNAGE.

Astrid .... Atalanta . Atalanta . Aihos .... Baltic .... 8. Banshee .
Banshee ... Barbara . Beata .... Beatrice . Beatrice . Bedouin .
Berolina . Bluebell . Britannia. 8. Bugey .... 8. Bullfinch . Capella....
Caracal.... Catalan.... Chiquita . Chita .... 8. Constance. Constance 8.
Consuta . 8. Coot Daisy .... Daisy .... Darling . Dinah .... Dizzy Dolly
Dolphin . Don 16 to 10 tons. 8. Dorian Doris Dotterel 8. Dragon Ply ..
9. Edith Elna Else Emerald Em'ly Emma 8. Emma Epervier Erin Ero
Esmeralda .. 8. Etna Ette 8. Eva Fedora 8. Fern Flavia 8. Fleohe
Fleur-de-Lys Flora Florence Folly Forerunner .. Freya II Fri Q-annet
Guzellen Geisha Gtem Gertrud 16 to 10 tons. 16 to 10 tons. Gioyanni
Gitana Gladys 9. Guy Hadassah Hadgie Hathor Hazy Morn Heidi
Hermione Herreshoff Hesperus Hirondelle 8. Hornet 8. Humming
Bird ... 8. Ibis 8. Ibis Iduna lemia Use Isabel Isea Kathleen Kestrel 8.
Kitty Kotiri Kraka 8. Lady of the Lake. . . L'Hirondelle Leontine
Limmershln Little Nell ' Mabel Magdalena I Maimuna Manxman ; 9.
Maple Leaf (The). I 8. Maria j 8. Marshlight i May I Medea 1 Mercury
I Merle I Merlin I Milady I Mimosa ' Minnie ' Momerath I Nanta I
Napoule Nautilus Nereda Ngaira Ninina No Name Nora 8 Noma
Oihonna Orang Outang .. Pastime Phantom Phyllis Piccolo Pierrette
Quickstep Raven Reine Restless 16 to 1 R§ve Reverie , 8. Roma
Romp Rothion Saianara St. Helen Saiph Samoa Shalimar Siola Siren
8kum Smew , Smut Spindrift Spinio Spinaway Starlight Stortebeoker
8. Swallow Talisman Tamaris Taniwha Teal VioU Violet Violet Vivien
Volador Wave Westralia White Wings Wild Rose ... Witta Wladimir
Zaph Zephyr Zuleika 10 to 6 tons. Digitized by 1199 Google
The text on this page is estimated to be only 25.90%
accurate

YACHT REGISTER. Jg ■ 10 to 5 tons. Barbara 10 9. Blanche

10 Bine Sea II 10 Bonafide 10 3. Bonaventure 10 Br:iz III 10 Brls 10
». Brownie 10 Brunehilde 10 Brunette 10 Brunette 10 Brynhild 10
Buzzy^Uoo 1<> I Cap.Ua 10 ' CaraMia 10 s. Carmen JO 8. Chilwa
10 Chrysalis \^ Ctirysanteme ^^ Ciss ,^ Constanter j^ Conauelo JJr
B. Crocodile J" Cuckoo... \^ Cuckoo II JO Culwalla 10 Cvane \^
Cyclone JO D 10 ViigaiAr 10 Daginar 10 Daland 10 Diana 10 P-" —
10 10 t IDora 10 Dora 10 Doris 10 I 8. Drone 10 | 8. Duchess of
Connaught 10 s. Ebor 10 I s. Echo 10 Elfvan 10 Ellen 0 ' EUoe 0 8.
Eneid 0 Epervier { ' s. Ethel 0 Ettarre Jo Evadne 10 9. Fairy Jp I
Falko 10 ' s. Faujfh-a-Balajrh... 10 Fearnousrht 10 , Firecrest 10
Firefly 10 ' I Fram 10 | I Freia 10 i 8. Frolic 10 Frolic 10 I Frolic 10 I
8. Frondeur (Le) 10 Fusion 10 I I Gabrielle 10 ' Gipsy lu , ! Gladys 10
; Gleam 10 I Glimt 10 I Gloriana 10 8. Glow-worm 10 Glunten 10
Godiva 10 Gondola 10 Good Luck 10 Gracie M 10 8. Guayracd *- 10
Gwendoline 10 Gweneth 10 Gyp 10 Halbran 10 Hazard 10
HeartseaKa 10 Hero 10 Hertog Hendrik ... 10 Hilda 10 Hollandia 10
Hypatia 10 Ibex 10 Idalia 10 lo 10 Irmgard 10 Isca 10 Iverna 10
Ixion 10 Jack Heron 10 Jeannette 10 Jenny 10 Jess 10 Karen
Johanne ... 10 Kelpie 10 Kingfisher 10 Kormoran 10 Lady Bird 10
Lansquenet 10 Laura 10 Laura 10 Lehetellen 10 Leonie 10 Lesmona
II 10 Lethe 10 Lily 10 Linda 10 H. Little Sabrina 10 a. Little See - me
• not 1 0 Lollypop 10 Lorielie 10 Lucifer 10 Luisilla 10 Magali 10
M^da 10 Magdalena 10 Mamba 10 Margot 10 Marie 10 I Marina 10 I
Mariska 10 ' Marjorie 10 Marmadyn 10 Maude 10 8. Mayflower 10
Maysie 10 Mercedes 10 Meteor 10 Mimine 10 Moray 10 1 Mouette 10
1200 ons. f 8. Muokel 10 B s. Muriel 10 Nami 10 Narval 10 S.Nina 10
Nirvana 10 s Nona 10 8. Noordster 10 i s Norveshets 10 i Oak Apple
10 a Oem 10 , s Olga 10 Oncle 10 Petronel 10 s Pilot 10 I Pilote 10
Primrose 10 Prisca 10 ; 8. Psyche 10 Puffin 10 Rambler 10 Ran 10 ;
Riva 10 ' 8. Robin 10 | Rose 10 I Royan 10 Runa 10 ' e Runa 10
Saida 10 San Toy 10 Sapphire 10 Schwan 10 | a Sea Breeze 10 Sea
Dog 10 | Digitized by LnOOQlC
The text on this page is estimated to be only 26.55%
accurate

LIST OF YACHTS ARRANGED ACCORDING TO TONNAGE.

10 to 6 tons. 10 to 6 tons. Vigilant 10 I Violet 10 I Virginia 10
Wanderer 10 Water Lily 10 WaterUly 10 I White Kitten 10 White
Wing 10 I Wieeel 10 ' WndDuck XO e. Wild Roae 10 ' Wild Rose 10
Winogene 10 Witch 10 Yvonne 10 Zephyr 10 (Ketch not named) 10 8
Aileen 9 | AlhatroB 9 Alfen 9 AUoe 9 Aluna 9 Alwida 9 AJyn 9 Amaoe
9 Amelita 9 Ami 9 8. Amy 9 Anker 9 B.Anna 9 Anna 9 Aotea 9
Argoey 9 Armorel 9 10 to 6 tons. Asra 9 8. Atlas 9 Ayah 9 Bangle 9
Beaver 9 Bijon 9 Black Duck 9 I Black TuUp 9 | Bonita 9 \ Brifle 9 i
Bmnehilde 9 Carmen 9 | Challenge 9 I Chemb 9 | Cinderella 9 Clytie
9 I Cock-a-too 9 i . Coey 9 I Conger 9 ; Coquette 9 i Cora 9 Coral 9 I
Cormorant 9 | Connani 9 i 8. Cymbeline 9 | s. Cymbula 9 , Dagny 9 '
Daisy II 9 • Daisy Bell 9 Daphne 9 8. . Dart 9 Darthnla 9 i Darthula 9
' Delight 9 10 to 5 tons. I 10 to 6 tons. Dianeme 9 Diavolo 9 Dodo 9
Donata 9 Doris 9 Elektra 9 ' Elfe 9 ; Ella 9 I Elna 9 i Elsie 9 I Enid 9
Eol 9 I Erling 9 j Ernestine • 9 | Espada 9 Estrella 9 Etincelle 9
Evame 9 Excelsior 9 Excelsior 9 Fairy Ella 9 Fal , 9 Fanny 9 Favorite 9
Fawn 9 Feltie 9 Fern 9 Filou 9 Fiona 9 Firefly 9 Flamingo 9 Flaneur 9
Flatfish 9 Fleur-de-Grdve 9 Flevo 9 j Fleur de Lis 9 Flip 9 Florence 9
Fram 9 Frimousse 9 Fusee 9 Gladys 9 Grayling 9 Greyfriar 9 Gudrun
9 Gwendolen 9 Gwenet^ 9 Gyptis 9 Halcyone 9 Hamilton 9 Hans 9
Hazard 9 B. Hibemia 9 Hirta 9 Hofifnung 9 Hoopoe 9 Hydra 9 Insula
9 Intrepid 9 lona 9 Iris 9 Istmo 9 Iziar 9 j s. Jeannette 9 > Joan 9 '
Jolantall 9 | Kaffir 9 I Karma 9 i Rennet 9 Kerenza 9 KingCoel 9 8.
Kittiwake 9 La Sir^e 9 Lady Madge (The) 9 Lahloo 9 Lamb 9 Latona
9 8. Leda 9 L6da 9 Lilian 9 Lilly 9 8. LUy 9 8. Little Prince 9 Llygra 9
Loma 9 Loven 9 Manon 9 Maigaret 9 Margaret 9 Margarethe 9
Margarethe 9 Margit 9 Marguerite- 9 Marjorie 9 Matador 9 Maudie 9
Mavis 9 8. May .-. 9 Merle 9 Mermaid 9 Meteor 9 Mica 9 10 to 6 tons.
Mignion 9 Mist 9 Moeel 9 s. Mowe 9 Moya 9 Myrtle 9 Mystery 9
Nance 9 Nancy 9 Natica 9 NelUe 9 Nemere 9 Nepenthe 9 Neptun 9
Ninja 9 Nokken 9 Nona 9 Nord 9 Norka 9 8. Oleggio Castello ... 9
Ondina 9 Oriel 9 8. Otter 9 s. Otto... 9 Pansy 9 Pastime 9 8. Pearl 9
Pepita 9 8. Pet 9 Petite Vitesse 9 Petrel 9 s. Pi-Ouit 9 Pipi ;.... " Pirate
1201
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