Principles of Spectrophotometry 169

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Principles of
Spectrophotometry

Much of what we know about the photosynthetic apparatus

was learned through spectroscopy—that is, measurements of the
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interaction of light and molecules. Spectrophotometry is an
important branch of spectroscopy that focuses on the technique
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of measurement. Here we will examine four topics: Beer’s law,

the measurement of absorbance, action spectra, and difference
spectra.
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Beer’s Law
An essential piece of information about any molecular species
is how much of it is present. Quantitative measures of concentration
are one of the cornerstones of biological science. Of all the methods
that have been devised for measuring concentration, by far the
most widely applied is absorption spectrophotometry. In this
technique, the amount of light that a sample absorbs at a particular
wavelength is measured and used to determine the concentration
of the sample by comparison with appropriate standards or
reference data. The most useful measure of light absorption is the
absorbance (A), also commonly called the optical density (OD).
The absorbance is defined as A = log I0 / I where I0 is the intensity
of light that is incident on the sample and I is the intensity of light
that is transmitted by the sample.
The absorbance of a sample can be related to the concentration
of the absorbing species through Beer’s law:
A = ε cl
 170 Concepts in Plant Physiology

where c is concentration, usually measured in moles per litter;

l is the length of the light path, usually 1 cm; and e is a
proportionality constant known as the molar extinction coefficient,
with the units of litres per mole per centimetre.
The value of e is a function of both the particular compound
being measured and the wavelength. Chlorophylls typically have
an e value of about 100,000 L mol–1 cm–1. When more than one
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component of a complex mixture absorbs at a given wavelength,

the absorbances due to the individual components are generally
additive.

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Figure: Definition of absorbance. A monochromatic incident light

beam of intensity I0 traverses a sample contained in a cuvette of
length (l). Some of the light is absorbed by the chromophores in the
sample, and the intensity of light that emerges is I.

The Spectrophotometer
The absorbance is measured by an instrument called a
spectrophotometer. The essential parts of a spectrophotometer
include a light source, a wavelength selection device such as
a monochromator or filter, a sample chamber, a light detector,
and a readout device, usually also include a computer, which is
used for storage and analysis of the spectra. The most useful
machines scan the wavelength of the light that is incident on the
sample and produce, as output, spectra of absorbance versus
wavelength.
 Principles of Spectrophotometry 171
Mono-chromator Transmitted Recorder or
Light Prism Sample light Photodetector computer

฀(nm)
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Mono-chromatic incident light

Figure: Schematic diagram of a spectrophotometer. The instrument
consists of a light source, a monochromator that contains a wavelength
selection device such as a prism, a sample holder, a photodetector, and a
recorder or computer. The output wavelength of the monochromator can
be changed by rotation of the prism;
the graph of absorbance versus wavelength is called a spectrum.

Action Spectra
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The use of action spectra has been central to the development
of our current understanding of photosynthesis. An action spectrum
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is a graph of the magnitude of the biological effect observed as

a function of wavelength. Examples of effects measured by action
spectra are oxygen evolution and hormonal growth responses
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due to the action of phytochrome. Often an action spectrum

can identify the chromophore responsible for a particular light-
induced phenomenon. Action spectra were instrumental in the
discovery of the existence of the two photosystems in O2-evolving
photosynthetic organisms.
Some of the first action spectra were measured by T. W.
Engelmann in the late 1800s. Engelmann used a prism to disperse
sunlight into a rainbow that was allowed to fall on an aquatic algal
filament. A population of O2-seeking bacteria was introduced into
the system. The bacteria congregated in the regions of the filaments
that evolved the most O2. These were the regions illuminated by
blue light and red light, which are strongly absorbed by chlorophyll.
Today, action spectra can be measured in room-sized spectrographs
in which the scientist enters a huge monochromator and places
samples for irradiation in a large area of the room bathed by
monochromatic light. But the principle of the experiment is the
same as that of Engelmann’s experiments.
Schematic diagram of the action spectrum measurements by
T. W. Engelmann. Engelmann projected a spectrum of light onto
 172 Concepts in Plant Physiology

the spiral chloroplast of the filamentous green alga Spirogyra and

observed that oxygen-seeking bacteria introduced into the system
collected in the region of the spectrum where chlorophyll pigments
absorb. This action spectrum gave the first indication of the
effectiveness of light absorbed by accessory pigments in driving
photosynthesis.
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Difference Spectra
An important technique in studies of photosynthesis is light-
induced difference spectroscopy, which measures changes in
absorbance. In this technique, bright light, often called actinic
light, is used to illuminate a sample, while a dim beam of light
is used to measure the absorbance of the sample at wavelengths
other than that of the actinic beam. In this way a difference spectrum
is obtained, which represents the changes in the absorption
spectrum of the sample induced by illumination with the actinic
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light. Absorption bands that disappear upon illumination appear
as negative peaks; new bands that appear upon illumination appear
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as positive peaks. Difference spectra give important clues to the

identity of molecular species participating in the photoreactions
of photosynthesis.
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By the use of special flash techniques, it is possible to record

the difference spectrum at a given time after flash excitation.
Multiple difference spectra recorded at different times after flash
excitation can be used to measure the kinetics of the chemical
reactions that follow photon excitation of a reaction centre. These
techniques can have extraordinary time resolution, in some cases
less than a picosecond (10–12 s), and have provided great insights
into the earliest events in the photosynthetic energy storage process.

Antagonistic Effects of Light on Cytochrome Oxidation

The puzzling red drop and enhancement effects were explained
by experiments performed by Louis Duysens of the Netherlands.
He suggested that two phtotchemical events were responsible for
enhancement effects. One photochemical event produced an
oxidation while the other produced a reduction. Chloroplasts
contain cytochromes, iron-containing proteins that function as
intermediate electron carriers in photosynthesis. Duysens found
that when a sample of a red alga was illuminated with long-
wavelength light, the cytochrome became mostly oxidized. If light
 Principles of Spectrophotometry 173

of a shorter wavelength was also present, the effect was partly

reversed. These antagonistic effects can be explained by a
mechanism involving two photochemical events: one that tended
to oxidize the cytochrome and one that tended to reduce it.
We know now that in the red region of the spectrum, one of
the photoreactions, known as photosystem I (PS-I), absorbs
preferentially far-red light of wavelengths greater than 680 nm,
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while the second, known as photosystem II (PS-II), absorbs red

light of 680 nm well and is driven very poorly by far-red light.
This wavelength dependence explains the enhancement effect and
the red drop effect. Another difference between the photosystems
is that Photosystem I produces a strong reductant, capable of
reducing NADP+, and a weak oxidant. Photosystem II produces
a very strong oxidant, capable of oxidizing water, and a weaker
reductant than the one produced by photosystem I. This reductant
of photosystem II rereduces the oxidant produced by photosystem
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I, which explains the antagonistic effect.
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Structures of Two Bacterial Reaction Centres

In 1984, Hartmut Michel, Johann Deisenhofer, Robert Huber,
and coworkers in Munich solved the three-dimensional structure
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of the reaction centre from the purple photosynthetic bacterium

Rhodopseudomonas viridis. This landmark achievement, for which
a Nobel prize was awarded in 1988, was the first high-resolution
X-ray structure determination for an integral membrane protein
and the first structure determination for a reaction centre complex.
The protein part of the complex consists of four separate
polypeptides. Two of them, called L and M (for light and medium
mass) bind all of the bacteriochlorophyll, quinone, and carotenoid
cofactors of the complex. The structure has a twofold symmetry
about an axis perpendicular to the plane of the membrane, hinting
at a dimeric nature of the reaction centre. The ten transmembrane
portions of the L and M peptides (five from each) are arranged
in α helices, and there are almost no charged amino acid residues
in the interior of the membrane. The H (heavy) protein has a single
transmembrane helix and is localized mostly on the cytoplasmic
side of the membrane. The C (cytochrome) subunit is located in
the periplasmic region (the region between the bacterial plasma
membrane and the outer membrane). The geometric arrangement
of the pigments and the quinones (electron acceptors), with the
 174 Concepts in Plant Physiology

protein removed. A similar arrangement is found in the reaction

centre of another purple photosynthetic bacterium, Rhodobacter
sphaeroides, except that the C subunit is not present.
Two of the bacteriochlorophyll molecules are in intimate
contact with each other and are known as the special pair. This
dimer, whose existence was predicted from magnetic-resonance
studies, is the photoactive portion of the complex. An electron is
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transferred from this dimer along the sequence of electron carriers

on the right side of the complex. Detailed analysis of these
structures, along with analysis of numerous mutants, has revealed
many of the principles involved in the energy storage processes
that are carried out by all reaction centres.
The bacterial reaction centre structure is thought to be similar
in many ways to that found in photosystem II from oxygen-
evolving organisms, especially in the electron acceptor portion of
the chain. The proteins that make up the core of the bacterial
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reaction centre are relatively similar in sequence to their
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photosystem II counterparts, implying an evolutionary relatedness.

Midpoint Potentials and Redox Reactions

Redox reactions, midpoint potentials, and their relationship
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to the laws of thermodynamics. These concepts are useful for our

discussion of electron flow from H2O to NADP+ and the interactions
between the different electron carriers.
The midpoint potential (Em) is a measure of the tendency of
a compound to take electrons from other compounds. A large positive
midpoint potential means that the compound is a strong oxidant;
a large negative value means that the compound is a strong reductant
(in both cases relative to the standard hydrogen electrode).
Equilibrium constants can easily be predicted from midpoint
potentials, in the same way that free energies were related to
equilibrium constants. Midpoint potentials for many chemical
and biochemical reactions have been measured and tabulated. The
y-axis on the Z scheme, midpoint potentials of the electron carriers,
with negative values higher than positive ones. This choice makes
reactions that are spontaneous (releasing free energy) appear
“downhill” on the graph.
Knowledge of the midpoint potentials of the various electron
carriers is important in establishing the pathway of electron flow
 Principles of Spectrophotometry 175

in any biochemical electron transport system, such as those found

in chloroplasts or mitochondria. Researchers make this
measurement usually by carrying out a redox titration. They adjust,
or poise, the sample at a particular redox potential, usually by
adding small amounts of oxidants or reductants. Redox mediators,
small molecules that permit rapid equilibration between the sample
and the electrodes of the measurement system, must be included
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to ensure that the system is at equilibrium when the measurement

is made. Several measurements are made at a variety of redox
potentials. The sample is stirred in a special cell that contains
platinum and reference electrodes, and chemical oxidants and
reductants are added to adjust the redox potential, which is read
with a voltmeter.
We can measure the extent of the redox reaction by following
a particular property of the sample, usually absorbance, at each
potential. In the example illustrated, the reduced form of the
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compound has an absorbance that decreases as the compound is
oxidized. The fraction of reduced form at each potential is plotted
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against redox potential, and the midpoint potential (E m) is

determined as the potential at which the compound is half oxidized
and half reduced.
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Oxygen Evolution
The chemical mechanism of photosynthetic water oxidation
is not yet known, although there is a great deal of indirect evidence
about the process. If a sample of dark-adapted photosynthetic
membrane is exposed to a sequence of very brief, intense flashes,
a characteristic pattern of oxygen production is observed. Little
or no oxygen is produced on the first two flashes, and maximal
oxygen is released on the third flash and every fourth flash
thereafter, until eventually the yield per flash damps to a constant
value. This remarkable result was first observed by Pierre Joliot
in the 1960s.
A schematic model explaining these observations, proposed
by Kok and coworkers, has been widely accepted. This model for
the photooxidation of water, called the S state mechanism, consists
of a series of five states, known as S0 to S4, which represent
successively more oxidized forms of the water-oxidizing enzyme
system, or oxygen-evolving complex. The light flashes advance
the system from one S state to the next, until state S4 is reached.
 176 Concepts in Plant Physiology

State S4 produces O2 without further light input and returns the

system to S0. Occasionally, a centre does not advance to the next
S state upon flash excitation, and less frequently, a centre is activated
twice by a single flash. These misses and double hits cause the
synchrony achieved by dark adaptation to be lost and the oxygen
yield eventually to damp to a constant value. After this steady
state has been reached, a complex has the same probability of
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being in any of the states S0 to S3 (S4 is unstable and occurs only

transiently), and the yield of O2 becomes constant. States S2 and
S3 decay in the dark, but only as far back as S1, which is stable in
the dark. Therefore, after adaptation to the dark, approximately
three-fourths of the oxygen-evolving complexes appear to be in
state S1 and one-fourth in state S0. This distribution of states explains
why the maximum yield of O2 is observed after the third of a series
of flashes given to dark-adapted chloroplasts.

Photosystem I
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The PS-I reaction centre is composed of a multiprotein complex.
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The reaction centre chlorophyll P700 and about 100 core antenna
chlorophylls are bound to two proteins, PsaA and PsaB, with
molecular masses in the range of 66 to 70 kDa. PS-I reaction centre
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complexes have been isolated from several organisms and found

to contain the 66 to 70 kDa proteins, along with a variable number
of smaller proteins in the range of 4 to 25 kDa. Some of these
proteins serve as binding sites for the soluble electron carriers
plastocyanin and ferredoxin. The functions of some of the other
proteins are not well understood. An 8-kDa protein contains some
of the bound iron-sulfur centres that serve as early electron
acceptors in photosystem I. The structure of the PS-I complex from
pea has been determined to a resolution of 4.4 E, and the positions
of many of the chlorophylls and electron transfer components
have been located.
In their reduced form, the electron carriers that function in the
acceptor region of photosystem I are all extremely strong reducing
agents. These reduced species are very unstable and thus difficult
to identify. Evidence indicates that one of these early acceptors is
a chlorophyll molecule, and another is a quinone species,
phylloquinone, also known as vitamin K1.
Additional electron acceptors include a series of three
membrane-associated iron–sulfur proteins, or bound ferredoxins,
 Principles of Spectrophotometry 177

also known as Fe–S centres Fe–SX, Fe–SA, and Fe–SB. Fe–S centre
Fe–SX is part of the P700-binding protein; centres Fe–SA and Fe–
SB reside on an 8 kDa protein that is part of the PS-I reaction centre
complex. Electrons are transferred through centres Fe–SA and Fe–
SB to ferredoxin, a small, water-soluble iron–sulfur protein. The
membrane-associated flavoprotein ferredoxin–NADP reductase
(FNR) reduces NADP+ to NADPH, thus completing the sequence of
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noncyclic electron transport that begins with the oxidation of water.

Electron transport between PSII and PSI is mediated by plastohydro-
quinone, the cytochrome b6f complex, and plastocyanin. The
cytochrome b6f complex contains two b-type hemes and one c-type heme.
A model for the organization of electron carriers in PS-I is
shown in Figure below. The P700 dimer is located at the bottom
of the structure and two symmetrical arms radiate from P700.
Each arm includes an accessory chlorophyll a and another
chlorophyll molecule tentatively identified as A0. Other structures
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include the Fe-S centre FX, and two other Fe-S centres, F1 and F2.
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The distance between the carriers is shown at the right.

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Figure: A model for the organization of electron

carriers in PS-I.
 178 Concepts in Plant Physiology

The PS-I reaction centre appears to have some functional

similarity to the reaction centre found in the anaerobic green
sulfur bacteria and the heliobacteria.
These bacteria contain low-potential Fe–S centres as early
electron acceptors and are probably capable of ferredoxin-mediated
NAD+ reduction similar to the NADP+ reduction function of
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photosystem I. There is almost certainly an evolutionary

relationship between these complexes and photosystem I of oxygen-
evolving organisms.

ATP Synthase
The chemical mechanism by which ATP is synthesized is not
yet understood in detail. However, considerable evidence now
supports a mechanism, first proposed by Paul Boyer, in which the
principal energy-requiring step is the release of bound ATP from
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the enzyme.
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Figure: The model of Boyer’s binding change mechanism.

The α and β subunits configure three nucleotide binding sites:
O, which provides the early binding site for ADP and inorganic
phosphate, L, to which the ADP and inorganic phosphate
bind after migrating from O, and T, which tightly binds ATP.
Energy ensuing from the movement of protons from the chloroplast
lumen to the stroma drives the rotation of the γ subunit of CF1, and the
interconversion of the binding sites and the release of an ATP molecule.
It has also been proposed that during catalysis a large portion
of the CF1 complex rotates about a bearing consisting of the γ
subunit. The γ subunit may act as a camshaft does, rotating
alternately against the α and α subunits. The energy of the
conformational movements is then translated into phosphoan-
hydride bond energy.
The thylakoid ATP synthase has two segments: CF0 is a
transmembrane segment, and CF1 is a hydrophilic segment at the
stomal surface. CF0 traslocates protons from the chloroplast lumen
 Principles of Spectrophotometry 179

to the catalytic part of the enzyme and CF1 cataylses the conversion
of ADP and inorganic phosphate to ATP.
The chloroplast ATP synthase has nine different subunits. CF1
is made of two large subunits, α and β, plus three smaller subunits,
γ, δ, and ε. Each CF1 molecule has three copies of the α and β
subunits and one copy of the γ, δ and ε subunits. The α and β subunits
bind ADP and phosphate and catalyize the phosphorylation of ADP
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into ATP.
Studies on the mitochondrial ATP synthase have increased
our understanding of how the ATP synthases work. As proposed
by Paul Boyer in 1997, ATP synthesis takes place by a binding
change mechanism. According to this model, the energy of the
proton gradient is used to release a tightly bound form of ATP
from a catalytic binding side of the enzyme. As shown in Figure
below, CF1 has three nucleotide binding sites, L, T and O. ADP
and inorganic phosphate are postulated to first bind to the O site,
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and then move to the L site. The T site binds ATP. As protons move
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from the lumen to the stromal region through the CF0 channel,
energy is released and the γ subunit of CF1 rotates. The rotation
causes conformation changes in the three nucleotide binding sites,
which interconvert, thus changing the afiity of the sites for the
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nucleotides. As the T site converts into a O site, ATP is released

and another cycle starts. Direct observation of the rotation of the
γ subunit of CF1 has provided strong experimental support for the
binding change mechanism.

Mode of Action of Some Herbicides

Herbicides of one major class—about half of the commercially
important compounds—act by interrupting photosynthetic electron
flow. A shows the chemical structure of two of these compounds.
The precise sites of action of many of these agents have been found
to lie either at the reducing side of photosystem I (for example,
in paraquat) or in the quinone acceptor complex in the electron
transport chain between the two photosystems.
Paraquat acts by intercepting electrons between the bound
ferredoxin acceptors and NADP and then reducing oxygen to
superoxide (O2–). Superoxide is a free radical that reacts
nonspecifically with a wide range of molecules in the chloroplast,
leading to the rapid loss of chloroplast activity. Lipid molecules
in cell membranes are especially sensitive.
 180 Concepts in Plant Physiology
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Paraquat
(methyl viologen)

DCMU (diuron)
(Dichlorophenyl-
dimethylurea)
Figure: The use of herbicides to kill unwanted plants is widespread in
modern agriculture. Many different classes of herbicides have been
developed, and they act by blocking amino acid, carotenoid, or lipid
biosynthesis or by disrupting cell division. Understanding the mode of
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action of herbicides has been an important tool in research on plant
metabolism and has facilitated their application under different
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agricultural practices (Ashton and Crafts 1981). Here, the chemical

structure of two herbicides that block photosynthetic electron flow are
shown. DCMU is also known as diuron. Paraquat has acquired public
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notoriety because of its use on marijuana crops.

The herbicides that act on the quinone acceptor complex
compete with plastoquinone for the QB binding site. If herbicide
is present, it displaces the oxidized form of plastoquinone and
occupies the specific binding site for the quinone acceptor, which
is thought to lie on the D1 herbicide-binding protein. The herbicide
is not able to accept electrons, so the electron is unable to leave
QA, the first quinone acceptor. Thus, the binding of herbicide
effectively blocks electron flow and inhibits photosynthesis. Many
herbicides that act in this manner also inhibit electron flow in
photosynthetic bacteria that have quinone-type electron acceptor
complexes.
In recent years, herbicide-resistant biotypes of common weeds
have appeared in areas where a single type of herbicide has been
used continuously for several years. These biotypes can be orders
of magnitude more resistant to certain classes of herbicides than
the nonresistant plant is. In several cases the resistance factor has
been traced to a single amino acid substitution in the D1 protein.
 Principles of Spectrophotometry 181

This change, presumably in the quinone (and herbicide) binding

region of the peptide, lowers the binding affinity of the herbicide,
making it much less effective.
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Figure: Sites of action of herbicides. Many herbicides, such as DCMU,

act by blocking electron flow at the quinone acceptors of photosystem II,
by competing for the binding site of plastoquinone that is normally
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occupied by QB. Other herbicides, such as paraquat, act by accepting

electrons from the early acceptors of photosystem I and then reacting
with oxygen to form superoxide, O2–, a species that is very damaging to
chloroplast components, especially lipids.
The possibility of fine-tuning the herbicide sensitivity of crop
plants by making subtle changes in the proteins of the PS-II reaction
centre has created a great deal of interest in the agricultural chemical
industry. Through biotechnology, it is now possible to make a crop
plant that is resistant to a particular herbicide, which can then be
applied to control undesirable plants that are not resistant. The
success of this approach will depend on whether undesirable side
effects of the herbicide-resistant mutations can be controlled and
on how rapidly the weeds acquire resistance through natural
selection or gene transfer. Thus, it is a race between science and
evolutionary change.
A complementary approach is to insert resistance factors to
certain pests, such as insects, into the plant via genetic engineering.
The plants then produce the toxic species themselves, eliminating
the need for insecticides.
 182 Concepts in Plant Physiology

Chlorophyll Biosynthesis
In the first phase of chlorophyll biosynthesis, the amino acid
glutamic acid is converted to 5-aminolevulinic acid (ALA). This
reaction is unusual in that it involves a covalent intermediate in
which the glutamic acid is attached to a transfer RNA molecule.
This is one of a very small number of examples in biochemistry
in which a tRNA is utilized in a process other than protein synthesis.
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Two molecules of ALA are then condensed to form porphobilinogen

(PBG), which ultimately form the pyrrole rings in chlorophyll. The
next phase is the assembly of a porphyrin structure from four
molecules of PBG. This phase consists of six distinct enzymatic
steps, ending with the product protoporphyrin IX.
All the biosynthesis steps up to this point are the same for the
synthesis of both chlorophyll and heme. But here the pathway
branches, and the fate of the molecule depends on which metal
is inserted into the centre of the porphyrin. If magnesium is inserted
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by an enzyme called magnesium chelatase, then the additional
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steps needed to convert the molecule into chlorophyll take place;

if iron is inserted, the species ultimately becomes heme.
The next phase of the chlorophyll biosynthetic pathway is the
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formation of the fifth ring (ring E) by cyclization of one of the

propionic acid side chains to form protochlorophyllide. The
pathway involves the reduction of one of the double bonds in ring
D, using NADPH. This process is driven by light in angiosperms
and is carried out by an enzyme called protochlorophyllide
oxidoreductase (POR). Non-oxygen-evolving photosynthetic
bacteria carry out this reaction without light, using a completely
different set of enzymes. Cyanobacteria, algae, lower plants, and
gymnosperms contain both the light-dependent POR pathway
and the light-independent pathway. Seedlings of angiosperms
grown in complete darkness lack chlorophyll, because the POR
enzyme requires light. These etiolated plants very rapidly turn
green when exposed to light. The final step in the chlorophyll
biosynthetic pathway is the attachment of the phytol tail, which
is catalyzed by an enzyme called chlorophyll synthetase.
The elucidation of the biosynthetic pathways of chlorophylls
and related pigments is a difficult task, in part because many of
the enzymes are present in low abundance. Recently, genetic
analysis has been used to clarify many aspects of these processes.