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Metal-Triggered FAD Reduction in D‑2-Hydroxyglutarate

Dehydrogenase from Pseudomonas aeruginosa PAO1
Joanna Afokai Quaye and Giovanni Gadda*
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ABSTRACT: Alcohol oxidation is an indispensable chemical

reaction in biological systems. This process, biologically catalyzed
by alcohol dehydrogenases (ADHs) and alcohol oxidases (AOXs),
follows two distinct chemical routes depending on the cofactor.
ADHs have been widely demonstrated to require Zn2+- and
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NAD(P)+-based cosubstrates. Except for galactose oxidase, AOXs

achieve their conversion of alcohols to aldehydes or ketones using
flavin-based cofactors. The FMN-dependent α-hydroxy acid-
oxidizing enzymes and the glucose−methanol−choline (GMC)
superfamily abstract their substrate’s α−OH proton using a
catalytic histidine, leading to substrate oxidation and flavin
reduction. However, there is no known alcohol oxidation
mechanism for enzymes requiring both a flavin and a metal. The Pseudomonas aeruginosa D-2-hydroxyglutarate dehydrogenase
(PaD2HGDH) is a recently characterized α-hydroxy acid dehydrogenase that converts D-2-hydroxyglutarate or D-malate to 2-
ketoglutarate or oxaloacetate, respectively. PaD2HGDH requires FAD and Zn2+ for catalysis. Previous studies on PaD2HGDH have
identified a highly conserved active site histidine residue whose position is topologically conserved for catalytic bases in FMN-
dependent α-hydroxy acid-oxidizing enzymes and the GMC superfamily of oxidoreductases. In this study, solvent isotope effects
(SIEs) coupled with pL-rate profiles and a viscosity control have been used to probe the role of the Zn2+ cofactor in the C2−OH
oxidation of D-malate and flavin reduction of PaD2HGDH. The data revealed an inverse solvent equilibrium isotope effect (SEIE) of
0.51 ± 0.09 consistent with a Zn2+-triggered abstraction of the substrate C2−OH proton that initiates D-malate oxidation and flavin
reduction. The system provides insights into the role of Zn2+ in the oxidation mechanism of PaD2HGDH and, by extension, metallo
flavoprotein dehydrogenases.
KEYWORDS: Pseudomonas aeruginosa D-2-hydroxyglutarate dehydrogenase, solvent isotope effects, viscosity effect, Zn2+,
metallo flavoprotein, flavin

■ INTRODUCTION
Alcohols are diverse molecules with unique chemical proper-
functional carbon, fragment to yield molecules with new
functional groups under harsh conditions.1
ties and versatile reactivity, making them indispensable in Alcohol oxidation is an essential and indispensable chemical
chemistry, biology, pharmacy, and industry.1 The versatile reaction central to metabolic pathways like glycolysis and
oxidation of alcohols to aldehydes, ketones, carboxylic acids, or gluconeogenesis, the Krebs cycle, xenobiotic metabolism, and
esters using oxidants like AgNO3, K2CrO4, or KMnO4 is a lipid metabolism in biological systems.4 Biocatalysis presents
fundamental transformation in organic chemistry, allowing for an environmentally friendly and renewable alternative to
the synthesis of several complex molecules. The mechanism of alcohol oxidation.5 Enzymes are selective catalysts, allowing
alcohol oxidation typically involves the formation of an precision chemistry without tedious protection group chem-
intermediate species, like a chromate ester or a transition istry.5 Most studies on alcohol oxidizing enzymes demonstrate
metal complex, followed by an elimination reaction or a the need for metals, flavins, or pyrroloquinoline quinone
rearrangement to form the oxidized product.1 The pathway of (PQQ) as cofactors for alcohol oxidation.4 For several decades,
alcohol oxidation primarily depends on the reaction conditions
and the availability of hydrogen atoms on the functional Received: October 3, 2024
carbon.2,3 Primary alcohols, having two hydrogen atoms on the Revised: November 21, 2024
functional carbon, are converted to aldehydes, with subsequent Accepted: November 25, 2024
oxidation to carboxylic acids. Secondary alcohols, having only Published: December 6, 2024
one hydrogen atom on the functional carbon, are oxidized to
ketones. Tertiary alcohols, lacking hydrogen atoms on the
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Scheme 1. General Reaction Mechanism of Alcohol Oxidation by Alcohol Oxidases and the FMN-Dependent α-Hydroxy Acid
Oxidizing Enzymesa

a
A conserved active site histidine abstracts a proton from the hydroxyl group, which triggers substrate oxidation and flavin reduction to yield the
carbonyl product and reduced flavin cofactor. In alcohol oxidases, R1 is either H or an alkyl group, and R2 is an alkyl group. In the FMN-dependent
α-hydroxy acid oxidizing enzymes, R1 is either H or an alkyl group, and R2 is a carboxylate group.

the catalytic mechanism of various alcohol oxidizing enzymes substrate oxidation and flavin reduction (Scheme 1). Similarly,
in biological systems has been investigated, including the the Glucose-Methanol-Choline (GMC) superfamily, sharing a
flavin-dependent vanillyl alcohol,6−9 aryl alcohol,10−16 and unique fold and a highly conserved active site, oxidizes alcohols
choline oxidases,4,17−20 nonheme iron-dependent oxy- using a highly conserved active site histidine, which abstracts a
genases,21 Cu(II)-dependent galactose oxidase,22 and the proton from the substrate OH group, initiating substrate
PQQ-dependent ethanol dehydrogenase.23 The newer PQQ- oxidation and flavin reduction (Scheme 1).28 There has been a
containing class of alcohol dehydrogenases employ an clear distinction between the ADH catalytic mechanism of
aspartate or glutamate for the alcohol OH proton abstraction, alcohol oxidation, which requires Zn2+ and NAD(P)+, and the
leading to PQQ reduction and substrate oxidation.23,24 The FMN-dependent α-hydroxy acid and GMC-type dehydrogen-
two major groups of enzymes commonly known to catalyze ase mechanism of alcohol oxidation, which requires a histidine
alcohol oxidation in biological systems are alcohol dehydro- and flavin cofactor. To date, a mechanism for alcohol oxidation
genases (ADHs) and alcohol oxidases (AOXs).25 While ADHs by an alcohol dehydrogenase with an active site histidine
have been widely demonstrated to require Zn2+ and NAD(P)+- requiring both a flavin-based cofactor and metal has not been
based cosubstrates, AOXs achieve their conversion of alcohols established.
to aldehydes or ketones using flavin-based cofactors.25 The Pseudomonas aeruginosa D-2-hydroxyglutarate dehydro-
Typically, a conserved active site histidine abstracts a proton genase (PaD2HGDH) is a recently characterized enzyme that
from the substrate OH group, triggering flavin reduction and converts the C2−OH in D-2-hydroxyglutarate to a carbonyl
formation of the carbonyl product in AOXs (Scheme 1).25 In group, yielding 2-ketoglutarate as a product.29−32 The enzyme
contrast, ADHs employ an active site metal to abstract the is also active with D-malate as an alternative substrate to yield
substrate OH proton, leading to NAD(P)+ reduction to form oxaloacetate (Scheme 3).31 PaD2HGDH plays a vital role in P.
NAD(P)H and substrate oxidation to yield the carbonyl
product (Scheme 2).26 Scheme 3. PaD2HGDH Catalytic Schemea

Scheme 2. General Reaction Mechanism of Alcohol

Oxidation by Alcohol Dehydrogenases (ADHs)a

a
n = 1 for D-malate and oxaloacetate. n = 2 for D-2-hydroxyglutarate
and 2-ketoglutarate.

aeruginosa by maintaining 2-ketoglutarate levels during

bacterial L -serine biosynthesis. 33,34 Knockout of the
PaD2HGDH gene has been demonstrated to inhibit the
a
An active site metal polarizes the hydroxyl group and is responsible growth of P. aeruginosa.33,34 PaD2HGDH has been shown to
for the OH proton abstraction. The OH proton is lost to an active site use FAD and Zn2+ as required cofactors (Figure 1), without
residue or the bulk solvent. The alkoxide formation triggers substrate which the enzyme is inactive.29,30 PaD2HGDH is different
oxidation and the reduction of the nicotine-based cosubstrate from other ADHs in that, although its physiological electron
NAD(P)+, yielding the carbonyl product and NAD(P)H. The circular acceptor is unknown, it does not use O2 as an electron
arrow used for the C2−OH group depicts a proton abstraction with an acceptor in the oxidative half-reaction.29,31 Previous studies on
unknown acceptor. Depending on the enzyme system, the proton PaD2HGDH have identified highly conserved amino acid
acceptor can either be the bulk solvent or an active site residue.
residues common to other α-hydroxy acid oxidizing enzymes.31
From a PaD2HGDH homology model built with SWISS-
Another group of alcohol-oxidizing enzymes is the FMN- MODEL using a putative dehydrogenase from Rhodopseudo-
dependent α-hydroxy acid-oxidizing enzymes that catalyze the monas palustris [Protein Data Bank (PDB) entry 3PM9] as a
oxidation of the α-hydroxy acid substrate to a keto acid template, these highly conserved amino acids are located in the
product.27 The catalytic mechanism of the FMN-dependent α- enzyme’s active site (Figure 1). A histidine residue is one such
hydroxy acid-oxidizing enzymes involves abstracting their conserved active site amino acid whose position is topologi-
substrate’s C2−OH proton using a fully conserved active-site cally conserved for catalytic bases in the FMN-dependent α-
histidine.28 The substrate OH proton abstraction triggers hydroxy acid and GMC-type superfamily of oxidoreductases.31
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substrate C2−OH upon substrate binding during enzyme

catalysis.29 Thus, the question remains whether the mechanism
of alcohol oxidation in PaD2HGDH follows the classical
FMN-dependent α-hydroxy acid/GMC-type catalysis requiring
an active site histidine and flavin (Scheme 1), ADH-type
catalysis requiring a metal (Scheme 2) or a new catalytic
process that is unique for a metallo flavoprotein, comprising a
combination of both mechanisms. With both FAD and Zn2+ as
cofactors and a conserved active site histidine, PaD2HGDH is
an excellent candidate for elucidating the catalytic mechanism
of a metallo flavoprotein alcohol dehydrogenase.
In the study described herein, solvent isotope effects (SIEs)
have been used to probe the role of the Zn2+ cofactor in the
Figure 1. Active site of PaD2HGDH showing the FAD and Zn2+ C2−OH oxidation of D-malate and flavin reduction of
cofactors and the conserved active site residues. The PaD2HGDH PaD2HGDH. The results provide insights into the oxidation
homology model was built with SWISS-MODEL using a putative mechanism of alcohols catalyzed by the Zn2+ and FAD-
dehydrogenase from Rhodopseudomonas palustris (Protein Data Bank dependent PaD2HGDH and, by extension, metallo flavopro-
code: 3PM9) as a template. The substrate D2HG and Zn2+ metal tein dehydrogenases with a GMC-type conserved active site
were obtained by the structural overlay of PaD2HGDH and its histidine.
human homologue (PDB 6LPP). The isoalloxazine ring of the FAD
cofactor is shown in gray; the Zn2+ cofactor is shown as a cyan sphere;
the substrate D2HG is shown in tan; the active site residues are
shown in brown; nitrogen atoms are shown in blue; oxygen atoms are
■ RESULTS
Rapid-Reaction Kinetics of PaD2HGDH with D-Malate as a
shown in red. The protein structures were visualized using UCSF Substrate
Chimera.36 To determine the rate of flavin reduction of the metallo
flavoprotein PaD2HGDH, the time-resolved anaerobic reduc-
Additionally, the homology model revealed a Zn2+ binding site tion of PaD2HGDH with D-malate was investigated under
comprising the flavin C4O atom and the fully conserved active pseudo-first-order conditions by monitoring the loss of
site amino acids H374, H380, and E420.30,31 The active site absorbance of the oxidized flavin at 450 nm at varying D-
topology of the PaD2HGDH homology model is similar to the malate concentrations from 0.2 to 150 mM. The resulting
active site of the published crystal structure of human stopped-flow traces showed 2 to 4 distinct reaction phases;
D2HGDH, which has fully conserved H434, H441, E475, and however, only the dominant phase with the largest magnitude
the flavin C4O atom as its Zn2+ ligands.31,35 depended on D-malate concentration. The stopped-flow traces
Previous PaD2HGDH studies have demonstrated a Zn2+- were fit with double, triple, or quadruple exponentials (eqs
mediated lowering of a Zn2+-hydrate pKa value to ∼7.0, 2−4) depending on the number of phases in the traces (Figure
yielding a Zn2+-hydroxide species in the ligand-free form of the 2A) to obtain the exponential decay rates for each phase (kobs1
enzyme.30 In the ligand-bound form, the water molecule to kobs4) at each substrate concentration. The UV−visible
coordinating the Zn2+ cofactor is replaced by the alcohol absorption spectrum at the end of each reaction showed
substrate. Given the Zn2+-mediated polarization of water in the characteristic properties of a fully reduced PaD2HGDH-bound
substrate-free enzyme, there is a likely polarization of the flavin, as previously reported, showing a charge transfer band

Figure 2. Anaerobic reduction of PaD2HGDH with D-malate as substrate. (A) Stopped-flow traces of PaD2HGDH at 450 nm with varying
concentrations of D-malate (0.2−150 mM) fit to eq 4. Note the log time scale. In the interest of clarity, 1 out of every 10 experimental points is
shown (vertical lines). Each trace is the average of triplicate runs at each substrate concentration. (B) Absorption spectra of PaD2HGDH showing
the fully oxidized flavin before reduction (Eox) and the fully reduced flavin after reduction with 150 mM of D-malate (Ered). (C) The dependence of
PaD2HGDH kobs1 on D-malate concentration fit to eq 5. The single point shown at each substrate concentration is the kobs value obtained from the
fit of the average of triplicate runs with eq 4, yielding an error of ≤5%. Data points for the kobs1 values in H2O are shown as ○, those in D2O are
shown as ●, and those in glycerol are shown as ⧫. Assays were carried out in 0.1 M ACES, 0.052 M Tris, 0.052 M ethanolamine, and 1 mM ZnCl2
using an SF-61DX2 Hi-Tech KinetAsyst high-performance stopped-flow spectrophotometer thermostated at 25 °C and equipped with a
photomultiplier detector under anaerobic conditions. The instrumental dead time is 2.2 ms. Only the representative data at pL 10.0, having the
glycerol control, are shown since all traces, spectra, and plots in protiated and deuterated buffered solutions across all pL values followed similar
trends.

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Table 1. Effect of pL on the Rapid-Reaction Kinetic Parameters of PaD2HGDH with D-Malatea

pL kred(H2O) (s−1) kred(D2O) (s−1) kred(Gly) (s−1) Kd(H2O) (mM) Kd(D2O) (mM) Kd(Gly) (mM)
10.0 680 ± 20 950 ± 90 490 ± 50 36 ± 3 67 ± 16 46 ± 13
9.5 570 ± 70 800 ± 40 20 ± 6 32 ± 4
9.0 360 ± 20 900 ± 50 5.0 ± 1.0 7.6 ± 1.2
8.5 340 ± 30 450 ± 30 3.6 ± 0.7 5.8 ± 0.9
8.0 200 ± 10 280 ± 10 3.1 ± 0.5 1.8 ± 0.4
7.5 100 ± 10 120 ± 10 3.4 ± 0.7 3.1 ± 0.7
7.0 33 ± 2 52 ± 3 2.9 ± 0.6 2.8 ± 0.6
6.5 7.5 ± 0.4 16 ± 1 3.7 ± 0.7 5.4 ± 0.7
6.0 1.6 ± 0.1 3.0 ± 0.1 0.7 ± 0.1 1.5 ± 0.2
a
Effect of pL on the rapid-reaction kinetic parameters kred and Kd of PaD2HGDH with D-malate. Each value is shown with the associated standard
error from fitting the kinetic data with eq 5. Assays were carried out in 0.1 M ACES, 0.052 M Tris, 0.052 M ethanolamine, and 1 mM ZnCl2 at 25
°C. D-Malate was varied from 0.2 mM to 150 mM, with enzyme concentration ranging from 5.5 μM to 7.3 μM to maintain pseudo-first-order
conditions.

between the Zn2+ cofactor and the reduced flavin (Figure The plot of the log kred values as a function of pL fit with eq
2B).29 6 showed an increase in the kred parameter from low to high pL
The dominant phase of the time-resolved anaerobic and plateaued above pL 9.0. The observed pKa value was 8.3 in
reduction of PaD2HGDH with D-malate, characterized by both protiated and deuterated buffered solutions, and the pL-
the bleaching of the oxidized flavin absorption at 450 nm, was independent limiting value (CH) at high pL was 600 s−1 in
assigned to flavin reduction. To obtain the limiting rate H2O and 930 s−1 in D2O (Figure 3 and Table 2).
constant for flavin reduction (kred), the exponential decay rates
for the dominant phase (kobs1) assigned to flavin reduction at
any given substrate concentration were fit with eq 5, yielding a
zero y-intercept hyperbolic dependence of the kobs1 parameter
on D-malate concentration (Figure 2C). The concentration at
which half the rate of flavin reduction is recorded (Kd) was also
determined from the curve.
pL Effects on the Rate of Flavin Reduction of PaD2HGDH
with D-Malate as a Substrate
To investigate the effect of pH on the PaD2HGDH flavin
reduction rate, the anaerobic reductive-half reaction assays
were carried out in buffered solutions from pH 6.0 to pH 10.0.
The resulting stopped-flow traces showed more reaction
phases with increasing pH. The data were fit with the Figure 3. pL dependence of the solvent isotope effects on the rate of
appropriate equations having the correct number of PaD2HGDH reduction with D-malate as substrate. Data points for
exponential terms corresponding to the number of phases in kred in H2O are shown as ○, and those in D2O are shown as ●. Values
the stopped-flow traces. At each pH, only the dominant phase were determined using the rapid-reaction kinetics approach with
depended on D-malate concentration, and there was a zero y- varying concentrations of D-malate as a substrate for PaD2HGDH
intercept hyperbolic dependence of the kobs1 parameter on D- between pL 6.0 and 10.0 under anaerobic conditions. The curves were
malate concentration after fitting the data with eq 5. The obtained by fitting the kinetic data to eq 6.
resulting kred and Kd kinetic parameters are reported in Table 1.
When the solvent was changed from H2O to D2O, and the Table 2. Properties of the pL Profile Plots of the Anaerobic
reaction was repeated in buffered solutions from pD 6.0 to pD Reduction of PaD2HGDH with D-Malatea
10.0 to investigate the solvent isotope effect on the rate of Property H2O D2O
flavin reduction, higher rates of flavin reduction were observed Limiting value (CH), s−1 600 ± 100 930 ± 110
across all pD values (Table 1). There were no observed pKa value 8.3 ± 0.1 8.4 ± 0.1
differences in the overall stopped-flow traces and enzyme a
Values were extrapolated from the pL profile plots obtained by fitting
spectra in the deuterated buffered solutions compared to the the kinetic data of the log kred parameter as a function of pL using eq 6
protiated buffered solutions. Additionally, there were no upon PaD2HGDH reduction with D-malate as a substrate between pL
significant differences in the values of the Kd parameter in 6.0 and 10.0 under anaerobic conditions.
protiated and deuterated buffered solutions between pL (pH
or pD) 6.5 and 9.0, with greater differences at the pL extremes.
Given that the kred kinetic parameter (k3) remains constant Effect of D2O Viscosity on the SIE of PaD2HGDH Flavin
above pL 9, the increased Kd parameter above pL 9 can be Reduction with D-Malate
explained as either a decrease in the rate constant of free To investigate the contribution of the higher D2O viscosity on
substrate and enzyme association to form the enzyme− the observed deuterium isotope effects on PaD2HGDH flavin
substrate (ES) complex (k1) or an increase in the rate constant reduction, a control viscosity experiment was carried out by
of substrate dissociation from the ES complex to yield free adding 9.67% glycerol, having the same relative viscosity as
substrate and enzyme species (k2). However, the data do not D2O, to all enzyme, buffer, and substrate solutions at pH 10.0.
allow for an unequivocal interpretation of the Kd pL profile. The control experiment was run at pH 10.0 since it falls in the
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pL-independent region of the plot of the log(kred) parameter were computed, yielding rate constants of ∼30 s−1 for kiso1, ∼ 3
against pL to ensure there was no pL effect on the observed s−1 for kiso2, and ∼0.5 s−1 for kiso3, irrespective of the solvent
isotope and viscosity effects. The data showed an effect of the being H2O or D2O (Table S1). Additionally, no viscosity effect
D2O viscosity on the solvent isotope effect measurement. was observed on the rate constants for the isomerization steps.
At pL 10.0, the fastest kred value was recorded in D2O,
yielding a limiting rate constant for flavin reduction of 950 s−1.
The second fastest kred was recorded in H2O with a value of
680 s−1, followed by the glycerol control having a kred value of
■ DISCUSSION
This study aimed to investigate the role of the PaD2HGDH
490 s−1 at pL 10.0 (Table 1 and Figure 2C). The isotope effect Zn2+ cofactor in the enzyme’s flavin reduction and the C2−OH
on the observed rate of flavin reduction measured in D2O oxidation of D-malate using solvent isotope effects coupled
DO with pL profile studies. When H2O is replaced with D2O in
( 2 k red) yielded a value of 0.71. However, the glycerol control, solvent isotope effect (SIE) studies, three outcomes are
having the same relative viscosity as D2O, yielded a normal expected: (i) an altered equilibrium of ionizable groups
viscosity effect (Glykred) of 1.38, consistent with an under- involved in enzyme catalysis upon replacing H2O with D2O.
estimation of the SIE arising from partial masking of the This altered equilibrium is observed as an altered pKa value in
isotope effect by the viscosity effect of the deuterated buffers. D2O compared to H2O, (ii) a viscosity effect arising from the
Thus, the actual value for the SIE (D2O *k red) is given by the higher viscosity of D2O than that of H2O, and (iii) a solvent
ratio of the isotope effect value (D2Ok red) to the glycerol effect isotope effect arising from the isotopic substitution of the
hydrogen in H2O with the deuterium in D2O.37,39−42 The SIE
value (Glykred), yielding a corrected SEIE D2O *k red value of 0.51
could either be a primary kinetic isotope effect (PKIE) or a
(Table 3). The computed inverse SIE is comparable to the solvent equilibrium isotope effect (SEIE). A PKIE originates
from the cleavage of the OH bond and arises from the
Table 3. Solvent Isotope Effect on the Rate of Flavin weakened donor-H/D bond in the transition state, yielding a
Reduction in PaD2HGDH with D-Malatea normal effect, i.e., a PKIE > 1; a SEIE originates from the
D2O
k red Gly
kred D2O *
k red
fractionation factors of the species in equilibrium with the H/
D and arises from the relative isotopic populations between
0.71 ± 0.08 1.38 ± 0.17 0.51 ± 0.09
two ground states in equilibrium, yielding either normal or
a
The solvent isotope effect (D2Ok red) was calculated by dividing the inverse effects, i.e., SEIE < 1.37 To determine the absolute
kred kinetic parameter obtained in H2O by that obtained in D2O. The contribution of the deuterium isotope on the kinetic
glycerol effect (Glykred) was calculated by dividing the kred kinetic measurement, conclusions must only be drawn in the pL-
parameter obtained in H2O by that obtained in H2O with 9.67% independent region of the SIE pL profile, which is devoid of pL
glycerol. The corrected solvent isotope effect (D2O *k red) is the given effects.37 Additionally, a viscosity control must be carried out
by the ratio of the isotope effect value (D2Ok red) to the glycerol effect to rule out any viscosity effects from replacing H2O with
value (Glykred). The standard deviation for each value was calculated D2O.37,38 The SIE data from this study demonstrate that Zn2+
by multiplying the value determined for the experimental parameter abstracts the C2−OH proton from D-malate and triggers flavin
by the square root of the sum of the squares of the percent errors reduction during substrate oxidation catalyzed by
contributed by each experimental parameter. PaD2HGDH. Moreover, an internal isomerization precedes
flavin reduction during PaD2HGDH catalysis. Details on the
Zn2+-triggered flavin reduction and D-malate oxidation
values reported for other metalloenzymes like thermolysin, mechanism in PaD2HGDH are discussed below.
carbonic anhydrase, and yeast alcohol dehydrogenase that
require metals for substrate−OH proton abstraction.37,38 The Zn2+ Cofactor Is Responsible for the C2−OH Proton
The three D-malate concentration-independent phases (kobs2, Abstraction during PaD2HGDH Substrate Oxidation
kobs3, and kobs4) in the stopped-flow traces during PaD2HGDH Evidence to support this conclusion comes from the solvent
flavin reduction might be due to multiple enzyme−substrate isotope studies on the anaerobic rapid reaction kinetics of
complex species interconverting and not in rapid equilibrium, PaD2HGDH with D-malate as a substrate. The results
with only one EoxS species being competent to yield the EredP presented in Figure 3 and Tables 2 and 3 and show the
complex. The alternative explanation that the other three presence of an inverse SIE. Due to the inverse nature of the
phases result from damaged enzyme species during the SIE on the rate of flavin reduction, a PKIE can be ruled out.
reaction preparation could not be ruled out, although it Thus, the observed inverse SIE on the kred parameter of
seemed less likely as typically, in our hands, flavin-dependent PaD2HGDH, having a value of 0.51 after correction for the
enzymes subjected to anaerobic flavin reduction yield at the viscosity effect in D2O, is consistent with a SEIE (given by the
most up to 10% of the enzyme being damaged in the ratio of the reactant fractionation factor, ϕ, to the product
experiment setup. fractionation factor) in which there is a Zn2+-mediated
Due to the lack of a dependence of the kobs2, kobs3, and kobs4 abstraction of the C2−OH proton, which triggers flavin
values on D-malate concentration, the rate constants of the reduction during PaD2HGDH catalysis of D-malate. Consid-
PaD2HGDH isomerization steps kiso1, kiso2, and kiso3 values ering that the catalytic step of flavin reduction is partially rate-
were computed from the averages of all the kobs2, kobs3, and kobs4 limiting in PaD2HGDH and the enzyme’s reverse commit-
values, respectively, across all D-malate concentrations at each ment to catalysis is non-negligible,29 an inverse SEIE is
pL value tested (Table S1). There was no observed SIE on the expected for a Zn2+-mediated substrate C2−OH deprotona-
various kiso values when the solvent was changed from H2O to tion, which is required for flavin reduction. Conventionally,
D2O, and there was no significant change in each kiso value solvent isotope effects coupled with pL profile studies
upon varying the pL from 6.0 to 10.0 (Table S1). Therefore, demonstrate the roles of catalytic metals in enzymes.37,39−41,43
the averages of the kiso1, kiso2, and kiso3 values across all pLs An observed inverse SIE would establish the presence of a
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Scheme 4. Proposed Mechanism of PaD2HGDH Flavin Reduction and D-Malate Oxidationa

a
The binding of D-malate displaces the active site Zn2+-hydrate/hydroxide. Zn2+ abstraction of the D-malate C2−OH proton triggers flavin
reduction and D-malate oxidation to yield oxaloacetate. For simplicity, the C2−OH proton abstraction and hydride transfer are shown in one step;
however, the data do not demonstrate whether these processes are concerted or sequential. The circular arrow used for the water OH and substrate
C2−OH groups indicates that the proton acceptor is unknown. For PaD2HGDH, the proton acceptor is likely an active site residue.

metal-hydroxide/alkoxide arising from the rapid-equilibrium chain is protonated at low pL values and cannot accept the
transfer of a metal-polarized proton in a rate-limiting step Zn2+-abstracted C2−OH proton, disfavoring catalysis. At high
during catalysis.37,40,43,44 Due to the partial positive charge on pL values, the competent unprotonated form of the side chain,
oxygen, when water or alcohol (A) is coordinated to Zn2+, the which can accept the Zn2+-abstracted C2−OH proton, is
fractionation factors, ϕ, of the Zn2+-hydrate/alcohol (ϕA), favored and dominant, facilitating catalysis. In flavin-dependent
describing the preference of exchangeable groups OH or OD enzymes that catalyze alcohol oxidation reactions like choline
for D or H (eq 1), are inverse and the reactant ϕ is smaller oxidase, the unprotonated catalytic group has been assigned to
than that of the product.37 Thus, the observed inverse SEIE a highly conserved active site histidine that acts as a base
can be explained as a rapid dissociation of the Zn2+-hydroxide during substrate oxidation.19,58
and equilibration of the Zn2+-alcohol substrate that triggers the Studies on Zn2+-dependent metalloenzymes that require a
loss of the C2−OH proton upon D-malate binding to yield the nucleophilic Zn2+-hydroxide for catalysis, like carbonic
Zn2+-alkoxide, as shown in Scheme 4.43,45 anhydrase, have reported increasing limbs from low to high
A similar mechanism involving substrate polarization by pLs in the pL profiles of steady-state kinetic parameters,
Zn2+ was previously proposed for PaD2HGDH after observing characteristic of an unprotonated group, as observed for
a charge transfer band arising from Zn2+ polarization of a Zn2+- PaD2HGDH.59 In such Zn2+-dependent metalloenzymes, the
hydrate species to yield a Zn2+-hydroxide species in the ligand- unprotonated catalytic group has been assigned to the
free form of PaD2HGDH.29 However, the pL profiles of the nucleophilic Zn2+-hydroxide species, which remains bound in
kcat and kcat/Km kinetic parameters were bell-shaped, and the the presence of substrates during enzyme catalysis.59 In a
pKa values for the protonated and unprotonated groups were previous study on PaD2HGDH, an increasing limb from low
less than 2 pL units apart, preventing the determination of a to high pL was observed in the kcat/Km pL profile, which
pL-independent region. Thus, an SIE could not be computed reported on the ligand-free form of the enzyme during catalysis
to allow the assignment of Zn2+ as the proton abstractor.29,30 and was assigned to a Zn2+-hydroxide species in the absence of
In contrast, systems that use active site amino acids like substrates.29 In agreement with the PaD2HGDH published
histidine for proton abstraction, such as glucose oxidase, data, a Zn2+-hydrate species has been reported in the ligand-
pyranose-2-oxidase, choline oxidase, and cholesterol oxidase, free form of the published crystal structure of human
yield normal or no SIEs when there is a proton in flight D2HGDH. However, in PaD2HGDH, since the Zn2+-
involved in the transition state of the substrate−OH bond hydroxide is displaced upon substrate binding 29 and
cleavage or if the active site preorganization facilitates the considering that the kred parameter probes the ligand-bound
formation of an alkoxide intermediate before substrate binding, state of the enzyme, the identity of the unprotonated group in
respectively, since the C2−OD group yields a slower rate of the kred parameter pL profile being the Zn2+-hydroxide is ruled
flavin reduction in deuterated buffered solutions.4,40,46−57 out. A similar explanation involving solvent hydroxide species
[OD][H 2O] can be ruled out since that scenario would yield a continuous
A = log positively sloped line from low to high pL due to the ever-
[OH][D2 O] (1) increasing concentration of hydroxide species with increasing
pL. Alternatively, the fully conserved H421 residue in the active
An Unprotonated Group Is Required for PaD2HGDH site of the enzyme is suitably positioned in a geometry and
Oxidation of D-Malate distance that can allow it to act as a proton acceptora upon the
This conclusion is supported by the reductive half-reaction pL Zn2+-triggered proton abstraction from the substrate C2−OH
profiles of PaD2HGDH with D-malate as a substrate. The pL group (Figure 4). However, the present data cannot be used to
profile of the kred parameter yielded a plot with a + 1 slope for conclude the identity of the unprotonated group required for
the increasing limb from low to high pL. The data are PaD2HGDH flavin reduction.
consistent with the requirement of an unprotonated group with The observation of the same pKa value for the unprotonated
a pKa value of ∼8.3 during substrate oxidation catalyzed by group in the pL profiles of the PaD2HGDH kred parameter in
PaD2HGDH. The data favor the assignment of the pKa the protiated and deuterated buffered solutions can be
associated with flavin reduction to the ionization of an explained by a compensation of the inverse effect of producing
amino acid side chain of an active site residue. The side the Zn2+-hydroxide ion, having a ∼+0.5 pKa shift in D2O, by
209 https://doi.org/10.1021/acsbiomedchemau.4c00108
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ACS Bio & Med Chem Au pubs.acs.org/biomedchemau Article

■ CONCLUSIONS
The mechanism of PaD2HGDH substrate oxidation follows
neither the classical FMN-dependent α-hydroxy acid/GMC-
type catalysis requiring a flavin cofactor and an active site
histidine as the substrate OH proton abstractor (Scheme 1)
nor the ADH-type catalysis requiring a nicotine-based
cosubstrate and a metal as the substrate OH proton abstractor
(Scheme 2). Instead, PaD2HGDH presents a unique blend of
both catalytic processes, requiring Zn2+ for the substrate OH
proton abstraction, a proton acceptor, which is likely the side
chain of an active site amino acid residue, and a flavin cofactor
(Scheme 4). This study is the first to demonstrate a metal-
triggered flavin reduction mechanism in a flavin-dependent
enzyme. This mechanism may be unique for the new class of
metallo flavoproteins.
Figure 4. Active site of PaD2HGDH showing the distances and This study has elucidated the role of Zn2+ in the C2−OH
locations of FAD, D2HG, Zn2+, H421, and E420. The PaD2HGDH oxidation of D-malate during PaD2HGDH catalysis, providing
homology model was built with SWISS-MODEL using a putative the first catalytic mechanism for a metallo flavoprotein
dehydrogenase from Rhodopseudomonas palustris (Protein Data Bank dehydrogenase with a conserved active site histidine. The
code: 3PM9) as a template. The substrate D2HG and Zn2+ metal solvent isotope effects coupled with pL profile studies
were obtained by the structural overlay of PaD2HGDH and its demonstrate that Zn2+-mediates abstraction of the C2−OH
human homologue (PDB 6LPP). The isoalloxazine ring of the FAD proton that leads to flavin reduction and substrate oxidation.
cofactor is shown in gray; the Zn2+ cofactor is shown as a cyan sphere;
the substrate D2HG is shown in tan; the active site residues are
This mechanism differs from the established mechanism for
shown in brown; nitrogen atoms are shown in blue; oxygen atoms are similar enzymes harboring a conserved active site histidine that
shown in red. The protein structures were visualized using UCSF abstracts the substrate OH proton for substrate oxidation.28
Chimera.36 The study demonstrates that in PaD2HGDH, which has both
Zn2+ and histidine in the active site, Zn2+ is responsible for the
abstraction of the substrate C2−OH proton; however, the
the inverse fractionation factor of the Zn2+-bound substrate ability of H421 to gain a proton during catalysis remains a
alkoxide species. Upon binding the Zn2+ cofactor, the substrate question. Moreover, PaD2HGDH flavin reduction is gated by
C2−OH group becomes a strong acid, making it easily an internal isomerization of the enzyme−substrate complex.
ionizable. The enzyme uses the Zn2+ cofactor to stabilize the This study is the first to assign a catalytic role to an active site
substrate alkoxide during the formation of the substrate Zn2+ in a flavoprotein dehydrogenase. Considering that
carbonyl group.40 Thus, the substrate C2−OH proton likely PaD2HGDH is one of the first-reported Zn2+ and FAD-
undergoes a reaction-induced change in its fractionation factor, dependent metallo flavoproteins, the study provides an
resulting in an inverse fractionation factor ϕ of ∼0.5, as essential foundation for understanding the catalytic mecha-
previously reported for horse liver alcohol dehydrogenase.60 nisms of other D2HGDH homologues and any FAD-
Consequently, the positive Zn2+-−OL pKa shift in D2O is dependent metallo flavoproteins to be discovered in the
canceled by the inverse ϕ of the Zn2+-alkoxide, leading to a net future. Furthermore, the Zn2+-triggered substrate C2−OH
zero effect. The net-zero effect is observed as similar inflection proton abstraction in the presence of a conserved histidine
points in the pH and pD profiles, yielding similar pKa values in described in this study provides a basis for more in-depth
D2O and H2O as reported for PaD2HGDH in this study. studies on the catalytic roles of conserved active site histidine
residues to prevent their incorrect assignment as catalytic bases
An Internal Isomerization of the Enzyme−Substrate in complex enzyme systems.
Complex Precedes Flavin Reduction in PaD2HGDH
Evidence supporting this conclusion comes from the kinetic
solvent viscosity effects (KSVEs) on the reductive-half reaction
of PaD2HGDH at pL 10.0, which affected the kred parameter.
■ EXPERIMENTAL PROCEDURES
Materials. D-Malate was purchased from Alfa Aesar (Haverhill,
The data suggest that internal isomerization of the enzyme− MA). Deuterium chloride (99.5%) and deuterium oxide
substrate complex gates flavin reduction in PaD2HGDH since (99.9%) were from Cambridge Isotope Laboratories, Inc.
flavin reduction is a chemical step that does not involve (Andover, MA). Glycerol, ACES, ethanolamine, Tris, ZnCl2,
solvent-sensitive protein motions.61 Viscosity effects are and all other reagents were of the highest commercial purity.
generally known to have no effects on the chemical steps of Recombinant D-2-hydroxyglutarate dehydrogenase from Pseu-
catalysis.61 However, when protein motions gating flavin domonas aeruginosa strain PAO1 (PaD2HGDH) was expressed
reduction occur at rates proportional to the rate of solvent from plasmid pET20b(+) harboring the PA0317 gene, which
fluctuations, KSVEs are observed on the kred parameter.62 In was designed in-lab and purchased from GenScript. As
PaD2HGDH, flavin reduction is likely gated by enzyme described previously,30 the protein was purified to homoge-
conformational changes arising from substrate binding and the neity and stored in 25 mM NaPO4, 1 mM ZnCl2, pH 7.4.
C2−OH proton abstraction.63,64 Such gated flavin reduction
has been previously observed for the flavin-dependent
Neurospora crassa class II nitronate monooxygenase, and a
■ RAPID-REACTION KINETICS
To investigate the mechanism of substrate oxidation in the
similar gated electron transfer has been reported for the heme metallo flavoprotein PaD2HGDH, rapid kinetics in protiated
and molybdenum-dependent chicken liver sulfite oxidase.65,66 and deuterated buffered solutions was carried out on a
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ACS Bio Med Chem Au 2025, 5, 204−214
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thermostated SF-61DX2 Hi-Tech KinetAsyst high-perform- A = B1 exp( kobs1t ) + B2 exp( kobs2t ) + B3 exp( kobs3t )
ance stopped-flow spectrophotometer equipped with a photo-
multiplier detector. To maintain a constant ionic strength +C (3)
across all pL values (“L” from lyonium or lyate is used to
denote either H or D, depending on the context), the reaction A = B1 exp( kobs1t ) + B2 exp( kobs2t ) + B3 exp( kobs3t )
was followed in protiated or deuterated 25 mM Good’s buffer + B4 exp( kobs4t ) + C (4)
(0.1 M ACES, 0.052 M Tris, and 0.052 M ethanolamine)
supplemented with 1 mM ZnCl2 at 25 °C and pL 6.0 to 10.0. The resulting kinetic parameters of the reductive half-
All pH measurements in D2O were determined by adding 0.4 reaction were determined after fitting the exponential decay
units to the electrode reading to account for the D2O effect on rate of the dominant phase, which was assigned to flavin
the pH electrode. The rate of flavin reduction was measured by reduction, at various D-malate concentrations with eq 5. The
monitoring the decrease in absorbance at 450 nm that results equation defines a hyperbolic saturation of the enzyme with D-
from the quenching of the oxidized flavin species upon mixing malate, yielding a y-intercept value of zero. The data were fit
the enzyme with the substrate. The reductive half-reaction was with the KaleidaGraph software (Synergy Software, Reading,
monitored anaerobically under pseudo-first-order conditions, PA). Here, kobs1 represents the exponential decay rate for
where the enzyme concentration after mixing with the reducing the enzyme-bound flavin at any substrate concen-
substrate was ∼8 μM, and D-malate concentrations ranged tration (S). kred is the limiting first-order rate constant for flavin
from 0.1 to 150 mM. reduction at saturating substrate concentrations. Kd is the
For this experiment, PaD2HGDH was first passed through a concentration of substrate at which half the limiting rate of
desalting PD-10 gel filtration column preequilibrated with flavin reduction is measured and is given by (k2+k3)/k1. The
either protiated or deuterated buffered solutions at various pL same data was obtained when an equation that defines a
values. The resulting enzyme solution was loaded into a hyperbolic saturation with a finite y-intercept was used.
tonometer and subjected to a 25-cycle degassing procedure by
alternately flushing with oxygen-free argon and applying a k redS
kobs1 =
vacuum to render the enzyme solution anaerobic. The Kd + S (5)
anaerobic enzyme was subsequently mounted onto the
stopped-flow instrument, which had been subjected to an The effects of pL on the flavin reduction of PaD2HGDH
were investigated by plotting the log values of the kred in
overnight treatment with an oxygen scrubbing system
protiated and deuterated buffered solutions as a function of pL
composed of 5 mM glucose and 1 μM glucose oxidase in
using eq 6. The equation describes a curve that increases with
100 mM sodium pyrophosphate at pH 6.0 and room
increasing pL with a slope of +1 and a pL-independent limiting
temperature. Substrate solutions were prepared by dissolving
value (CL) at high pL.
D-malate in protiated or deuterated buffered solutions with
subsequent pL adjustments. The substrate solutions were then CL
loaded into syringes and flushed for 30 min with argon before log Y = log
10 pL
being mounted onto the stopped-flow instrument. Addition- (1 + 10 pKa ) (6)
ally, 2 mM glucose and 0.5 μM glucose oxidase were present in
all buffers, enzyme solutions, and substrate solutions to remove
traces of oxygen.
The effects of the isotopically labeled solvent on the rapid
■ ASSOCIATED CONTENT
Data Availability Statement
kinetics were determined by alternating solvent isotopomers. All data are contained within the manuscript except that
The enzyme and D-malate volumes were mixed in the stopped- enclosed in the Supporting Information.
flow spectrophotometer in single-mixing mode following
established procedures with an instrument mixing time of 2.2
*
sı Supporting Information

ms. The activity was assayed in triplicate for each substrate The Supporting Information is available free of charge at
concentration, and the average value was considered. Typically, https://pubs.acs.org/doi/10.1021/acsbiomedche-
measurements differed by ∼5%. mau.4c00108.
Isotope and pL effects on enzyme isomerization steps
■ DATA ANALYSIS (Table S1) (PDF)
The time-resolved flavin reductions at each pL in protiated and
deuterated buffered solutions were fit to eqs 2−4 depending on
the number of phases observed in the stopped-flow traces,
■ AUTHOR INFORMATION
Corresponding Author
which describes a quadruple exponential process for flavin Giovanni Gadda − Departments of Chemistry, Biology, and
reduction. Here, kobs1, kobs2, kobs3, and kobs4 represent the The Center for Diagnostics and Therapeutics, Georgia State
exponential decay rates for each phase during the reduction of University, Atlanta, Georgia 30302-3965, United States of
the enzyme-bound flavin at any given substrate concentration America; orcid.org/0000-0002-7508-4195;
at 450 nm. A represents the absorbance at 450 nm at any given Phone: (404) 413-5537; Email: [email protected];
time, B1, B2, B3, and B4 are the amplitudes of the absorption Fax: (404) 413-5505
changes, t is time, and C is the absorbance at an infinite time
that accounts for the nonzero absorbance of the fully reduced Author
enzyme-bound flavin. Joanna Afokai Quaye − Departments of Chemistry, Georgia
State University, Atlanta, Georgia 30302-3965, United States
A = B1 exp( kobs1t ) + B2 exp( kobs2t ) + C (2) of America
211 https://doi.org/10.1021/acsbiomedchemau.4c00108
ACS Bio Med Chem Au 2025, 5, 204−214
ACS Bio & Med Chem Au pubs.acs.org/biomedchemau Article

Complete contact information is available at: (6) Gygli, G.; de Vries, R. P.; van Berkel, W. J. H. On the Origin of
https://pubs.acs.org/10.1021/acsbiomedchemau.4c00108 Vanillyl Alcohol Oxidases. Fungal Genet Biol. 2018, 116, 24−32.
(7) Mattevi, A.; Fraaije, M. W.; Mozzarelli, A.; Olivi, L.; Coda, A.;
Author Contributions Van Berkel, W. J. H. Crystal Structures and Inhibitor Binding in the
Octameric Flavoenzyme Vanillyl-Alcohol Oxidase: The Shape of the
This manuscript was written with contributions from all Active-Site Cavity Controls Substrate Specificity. Structure 1997, 5
authors, and all authors have approved the final version of the (7), 907−920.
manuscript. (8) Fraaije, M. W.; Van Den Heuvel, R. H. H.; Van Berkel, W. J. H.;
Funding Mattevi, A. Structural Analysis of Flavinylation in Vanillyl-Alcohol
Oxidase. J. Biol. Chem. 2000, 275 (49), 38654−38658.
This work was supported by Georgia State University’s (9) Gygli, G.; Lucas, M. F.; Guallar, V.; van Berkel, W. J. H. The Ins
Department of Chemistry. and Outs of Vanillyl Alcohol Oxidase: Identification of Ligand
Notes Migration Paths. PloS Comput. Biol. 2017, 13 (10), No. e1005787.
(10) Serrano, A.; Carro, J.; Martínez, A. T. Reaction Mechanisms
The authors declare no competing financial interest. and Applications of Aryl-Alcohol Oxidase. Enzymes 2020, 47, 167−
192.
■ ACKNOWLEDGMENTS
We acknowledge the Resource for Biocomputing, Visual-
(11) Carro, J.; Martínez-Júlvez, M.; Medina, M.; Martínez, A. T.;
Ferreira, P. Protein Dynamics Promote Hydride Tunnelling in
Substrate Oxidation by Aryl-Alcohol Oxidase. Phys. Chem. Chem.
ization, and Informatics at the University of California, San Phys. 2017, 19 (42), 28666−28675.
Francisco, with support from NIH P41-GM103311, for the (12) Carro, J.; Ferreira, P.; Martínez, A. T.; Gadda, G. Stepwise
molecular graphics and analyses performed with UCSF Hydrogen Atom and Proton Transfers in Dioxygen Reduction by
Chimera. We thank Dr. Daniel M. Quinn, Dr. Paul F. Aryl-Alcohol Oxidase. Biochemistry 2018, 57 (11), 1790−1797.
Fitzpatrick, and Dr. Bryce V. Plapp for their insightful (13) Varela, E.; Jesús Martínez, M.; Martínez, A. T. Aryl-Alcohol
discussions. Oxidase Protein Sequence: A Comparison with Glucose Oxidase and
Other FAD Oxidoreductases. Biochim. Biophys. Acta Protein Struct.

■ ABBREVIATIONS
PaD2HGDH, Pseudomonas aeruginosa D-2-hydroxyglutarate
Mol. Enzym. 2000, 1481 (2000), 202−208.
(14) Hernández-Ortega, A.; Lucas, F.; Ferreira, P.; Medina, M.;
Guallar, V.; Martínez, A. T. Role of Active Site Histidines in the Two
dehydrogenase; D2HG, D-2-hydroxyglutarate; SIE, solvent Half-Reactions of the Aryl-Alcohol Oxidase Catalytic Cycle.
isotope effect; SKIE, solvent kinetic isotope effect; SEIE, Biochemistry 2012, 51 (33), 6595−6608.
solvent equilibrium isotope effect; KSVE, kinetic solvent (15) Fernández, I. S.; Ruíz-Duẽas, F. J.; Santillana, E.; Ferreira, P.;
viscosity effect. Martínez, M. J.; Martínez, Á . T.; Romero, A. Novel Structural
Features in the GMC Family of Oxidoreductases Revealed by the

■
a
ADDITIONAL NOTE
According to the IUPAC Gold Book, a base is a chemical
Crystal Structure of Fungal Aryl-Alcohol Oxidase. Acta Crystallogr.,
Sect. D: Biol. Crystallogr. 2009, 65 (11), 1196−1205.
(16) Mathieu, Y.; Piumi, F.; Valli, R.; Aramburu, J. C.; Ferreira, P.;
species with an available pair of electrons capable of forming a Faulds, C. B.; Record, E. Activities of Secreted Aryl Alcohol Quinone
covalent bond with a proton or with the vacant orbital of Oxidoreductases from Pycnoporus Cinnabarinus Provide Insights into
another species.67−69 The term “catalytic base,” which does not Fungal Degradation of Plant Biomass. Appl. Environ. Microbiol. 2016,
exist in the IUPAC Gold Book, was coined as a convenient way 82 (8), 2411−2423.
(17) Gadda, G. PH and Deuterium Kinetic Isotope Effects Studies
for enzymologists to describe a chemical species that can act on the Oxidation of Choline to Betaine-Aldehyde Catalyzed by
(i) as a catalyst by abstracting a proton during enzymatic Choline Oxidase. Biochim. Biophys. Acta, Proteins Proteomics 2003,
reactions and (ii) as a base by forming a covalent bond with 1650 (1−2), 4−9.
the abstracted proton. When acting as a catalytic base, a (18) Gadda, G. Kinetic Mechanism of Choline Oxidase from
histidine side chain doubles as the catalyst and the proton Arthrobacter Globiformis. Biochim. Biophys. Acta, Proteins Proteomics
acceptor. When alcohol proton abstraction is catalyzed by a 2003, 1646 (1−2), 112−118.
metal cofactor like Zn2+, the proton released by the OH group (19) Smitherman, C.; Rungsrisuriyachai, K.; Germann, M. W.;
requires an acceptor, which can be either the bulk solvent or an Gadda, G. Identification of the Catalytic Base for Alcohol Activation
active site residue like a histidine if the ES complex has in Choline Oxidase. Biochemistry 2015, 54 (2), 413−421.
geometric constraints shielding solvent access to the OH (20) Gadda, G. 8 Choline Oxidase and Related Systems; De Gruyter:
group. 2013, .
(21) White, M. D.; Flashman, E. Catalytic Strategies of the Non-

■ REFERENCES
(1) Khan, A. A. Advancement in Alcohol Chemistry: Synthesis,
Heme Iron Dependent Oxygenases and Their Roles in Plant Biology.
Curr. Opin Chem. Biol. 2016, 31, 126−135.
(22) Himo, F.; Eriksson, L. A.; Maseras, F.; Siegbahn, P. E. M.
Properties and Applications. Int. J. Res. Publ. Rev. 2024, 5, 1523− Catalytic Mechanism of Galactose Oxidase: A Theoretical Study. J.
1525. Am. Chem. Soc. 2000, 122 (33), 8031−8036.
(2) Mohrig, J. R.; Hammond, C. N.; Schatz, P. F. Techniques in (23) Kay, C. W. M.; Mennenga, B.; Görisch, H.; Bittl, R. Structure of
Organic Chemistry; 3rd Ed.; Macmillan, 2010. the Pyrroloquinoline Quinone Radical in Quinoprotein Ethanol
(3) Wang, F.; Stahl, S. S. Electrochemical Oxidation of Organic Dehydrogenase. J. Biol. Chem. 2006, 281 (3), 1470−1476.
Molecules at Lower Overpotential: Accessing Broader Functional (24) Matsushita, K.; Toyama, H.; Yamada, M.; Adachi, O.
Group Compatibility with Electron-Proton Transfer Mediators. Acc. Quinoproteins: Structure, Function, and Biotechnological Applica-
Chem. Res. 2020, 53 (3), 561−574. tions. Appl. Microbiol. Biotechnol. 2002, 58, 13−22.
(4) Fan, F.; Gadda, G. On the Catalytic Mechanism of Choline (25) Goswami, P.; Chinnadayyala, S. S. R.; Chakraborty, M.; Kumar,
Oxidase. J. Am. Chem. Soc. 2005, 127 (7), 2067−2074. A. K.; Kakoti, A. An Overview on Alcohol Oxidases and Their
(5) Puetz, H.; Puchl’ová, E.; Vranková, K.; Hollmann, F. Biocatalytic Potential Applications. Appl. Microbiol. Biotechnol. 2013, 97, 4259−
Oxidation of Alcohols. Catalysts 2020, 10 (9), 952. 4275.

212 https://doi.org/10.1021/acsbiomedchemau.4c00108
ACS Bio Med Chem Au 2025, 5, 204−214
ACS Bio & Med Chem Au pubs.acs.org/biomedchemau Article

(26) Milagre, C. D. F.; Milagre, H. M. S. Alcohol Dehydrogenase- (45) Karsten, W. E.; Lai, C.-J.; Cook, P. F. Inverse Solvent Isotope
Catalyzed Oxidation. Curr. Opin. Green Sustain. 2022, 38 (2022), Effects in the NAD-Malic Enzyme Reaction Are the Result of the
100694. Viscosity Difference between D2O and H2O: Implications for Solvent
(27) Furuichi, M.; Suzuki, N.; Dhakshnamoorhty, B.; Minagawa, H.; Isotope Effect Studies. J. Am. Chem. Soc. 1995, 117 (22), 5914−5918.
Yamagishi, R.; Watanabe, Y.; Goto, Y.; Kaneko, H.; Yoshida, Y.; Yagi, (46) Gadda, G. Hydride Transfer Made Easy in the Reaction of
H.; Waga, I.; Kumar, P. K. R.; Mizuno, H. X-Ray Structures of Alcohol Oxidation Catalyzed by Flavin-Dependent Oxidases.
Aerococcus Viridans Lactate Oxidase and Its Complex with d-Lactate at Biochemistry 2008, 47 (52), 13745−13753.
PH 4.5 Show an α-Hydroxyacid Oxidation Mechanism. J. Mol. Biol. (47) Sucharitakul, J.; Wongnate, T.; Chaiyen, P. Kinetic Isotope
2008, 378 (2), 436−446. Effects on the Noncovalent Flavin Mutant Protein of Pyranose 2-
(28) Romero, E.; Gadda, G. Alcohol Oxidation by Flavoenzymes. Oxidase Reveal Insights into the Flavin Reduction Mechanism.
Biomol. Concepts 2014, 5 (4), 299−318. Biochemistry 2010, 49 (17), 3753−3765.
(29) Quaye, J. A.; Gadda, G. The Pseudomonas Aeruginosa (48) Fan, F.; Gadda, G. An Internal Equilibrium Preorganizes the
PAO1Metallo Flavoprotein D-2-Hydroxyglutarate Dehydrogenase Enzyme-Substrate Complex for Hydride Tunneling in Choline
Requires Zn2+ for Substrate Orientation and Activation. J. Biol. Oxidase. Biochemistry 2007, 46 (21), 6402−6408.
Chem. 2023, 299 (3), 103008. (49) Kurtz, K. A.; Rishavy, M. A.; Cleland, W. W.; Fitzpatrick, P. F.
(30) Quaye, J. A.; Gadda, G. Uncovering Zn2+ as a Cofactor of FAD- Nitrogen Isotope Effects As Probes of the Mechanism of D-Amino
Dependent Pseudomonas Aeruginosa PAO1 D-2-Hydroxyglutarate Acid Oxidase. J. Am. Chem. Soc. 2000, 122 (51), 12896−12897.
Dehydrogenase. J. Biol. Chem. 2023, 299 (3), 103007. (50) Denu, J. M.; Fitzpatrick, P. F. Intrinsic Primary, Secondary, and
(31) Quaye, J. A.; Gadda, G. Kinetic and Bioinformatic Character- Solvent Kinetic Isotope Effects on the Reductive Half-Reaction of d-
ization of D-2-Hydroxyglutarate Dehydrogenase from Pseudomonas Amino Acid Oxidase: Evidence against a Concerted Mechanism.
Aeruginosa PAO1. Biochemistry 2020, 59 (51), 4833−4844. Biochemistry 1994, 33 (13), 4001−4007.
(32) Quaye, J.Biochemical Characterization of Selected Flavin- (51) Roth, J. P.; Klinman, J. P. Catalysis of Electron Transfer during
Dependent Metabolic Enzymes of Pseudomonas Aeruginosa PAO1 Activation of O2 by the Flavoprotein Glucose Oxidase. Proc. Natl.
Toward Alternative Therapeutic DevelopmentScholarlyworks @ Georgia Acad. Sci. U.S.A. 2003, 100 (1), 62−67.
State University2022https://scholarworks.gsu.edu/chemistry_diss/ (52) Amar, D.; North, P.; Miskiniene, V.; Cenas, N.; Lederer, F.
238 Hydroxamates as Substrates and Inhibitors for FMN-Dependent.
(33) Zhang, W.; Zhang, M.; Gao, C.; Zhang, Y. Y.; Ge, Y.; Guo, S.; Bioorg. Chem. 2002, 30, 145−162.
Guo, X.; Zhou, Z.; Liu, Q.; Zhang, Y. Y.; Ma, C.; Tao, F.; Xu, P. (53) Wohlfahrt, G.; Witt, S.; Hendle, J.; Schomburg, D.; Kalisz, H.
Coupling between D-3-Phosphoglycerate Dehydrogenase and D-2- M.; Hecht, H. J. 1.8 and 1.9 Å Resolution Structures of the Penicillium
Hydroxyglutarate Dehydrogenase Drives Bacterial L-Serine Synthesis. Amagasakiense and Aspergillus Niger Glucose Oxidases as a Basis for
Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (36), No. E7574−E7582.
Modelling Substrate Complexes. Acta Crystallogr. D 1999, 55 (5),
(34) Guo, X.; Zhang, M.; Cao, M.; Zhang, W.; Kang, Z.; Xu, P.; Ma,
969−977.
C.; Gao, C. D-2-Hydroxyglutarate Dehydrogenase Plays a Dual Role
(54) Vrielink, A.; Ghisla, S. Cholesterol Oxidase: Biochemistry and
in L -Serine Biosynthesis and Utilization in the Bacterium
Structural Features. Febs J. 2009, 276 (23), 6826−6843.
Pseudomonas Stutzeri. J. Biol. Chem. 2018, 293 (40), 15513−15523.
(55) Lehoux, I. E.; Mitra, B. (S)-Mandelate Dehydrogenase from
(35) Toplak, M.; Brunner, J.; Schmidt, J.; Macheroux, P.
Pseudomonas Putida: Mutations of the Catalytic Base Histidine-274
Biochemical Characterization of Human D-2-Hydroxyglutarate
and Chemical Rescue of Activity. Biochemistry 1999, 38 (31), 9948−
Dehydrogenase and Two Disease Related Variants Reveals the
9955.
Molecular Cause of D-2-Hydroxyglutaric Aciduria. Biochim. Biophys.
(56) Fitzpatrick, P. F. Substrate Dehydrogenation by Flavoproteins.
Acta, Proteins Proteomics 2019, 1867 (11), 140255.
(36) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Acc. Chem. Res. 2001, 34 (4), 299−307.
(57) Fitzpatrick, P. F. Carbanion versus Hydride Transfer
Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera - A
Visualization System for Exploratory Research and Analysis. J. Mechanisms in Flavoprotein-Catalyzed Dehydrogenations. Bioorg.
Comput. Chem. 2004, 25 (13), 1605−1612. Chem. 2004, 32 (3), 125−139.
(37) Fernandez, P. L.; Murkin, A. S. Inverse Solvent Isotope Effects (58) Wongnate, T.; Sucharitakul, J.; Chaiyen, P. Identification of a
in Enzyme-Catalyzed Reactions. Molecules 2020, 25 (8), 1933. Catalytic Base for Sugar Oxidation in the Pyranose 2-Oxidase
(38) Karsten, W. E.; Lai, C.-J.; Cook, P. F. Inverse Solvent Isotope Reaction. ChemBiochem 2011, 12 (17), 2577−2586.
Effects in the NAD-Malic Enzyme Reaction Are the Result of the (59) Mccall, K. A.; Huang, C.-C.; Fierke, C. A. Zinc and Health:
Viscosity Difference between D2O and H2O: Implications for Solvent Current Status and Future Directions Function and Mechanism of
Isotope Effect Studies. J. Am. Chem. Soc. 1995, 117, 5914−5918. Zinc Metalloenzymes 1. J. Nutr. 2000, 130 (5), 1437S−1446S.
(39) Cook, P. F.; Cleland, W. W. PH Variation of Isotope Effects in (60) Sekhar, V. C.; Plapp, B. V. Rate Constants for a Mechanism
Enzyme-Catalyzed Reactions. 2. Isotope-Dependent Step Not PH Including Intermediates in the Interconversion of Ternary Complexes
Dependent. Kinetic Mechanism of Alcohol Dehydrogenase. Bio- by Horse Liver Alcohol Dehydrogenase. Biochemistry 1990, 29,
chemistry 1981, 20 (7), 1805−1816. 4289−4295.
(40) Fitzpatrick, P. F. Combining Solvent Isotope Effects with (61) Gadda, G.; Sobrado, P. Kinetic Solvent Viscosity Effects as
Substrate Isotope Effects in Mechanistic Studies of Alcohol and Probes for Studying the Mechanisms of Enzyme Action. Biochemistry
Amine Oxidation by Enzymes. Biochim. Biophys. Acta Proteins 2018, 57 (25), 3445−3453.
Proteom. 2015, 1854, 1746−1755. (62) Romero, E.; Ladani, S. T.; Hamelberg, D.; Gadda, G. Solvent-
(41) Wallace Cleland, W. Use of Isotope Effects to Elucidate Slaved Motions in the Hydride Tunneling Reaction Catalyzed by
Enzyme Mechanism. CRC Crit. Rev. Biochem. 1982, 13 (4), 385−428. Human Glycolate Oxidase. ACS Catal. 2016, 6 (3), 2113−2120.
(42) Millero, F. J.; Dexter, R.; Hoff, E. Density and Viscosity of (63) Francis, K.; Russell, B.; Gadda, G. Involvement of a
Deuterium Oxide Solutions from 5−70° C. J. Chem. Eng. Data 1971, Flavosemiquinone in the Enzymatic Oxidation of Nitroalkanes
16 (1), 85−87. Catalyzed by 2-Nitropropane Dioxygenase. J. Biol. Chem. 2005, 280
(43) Schmidt, J.; Chen, J.; Detraglia, M.; Minkel, D.; Mcfarland, J. T. (7), 5195−5204.
Solvent Deuterium Isotope Effect on the Liver Alcohol Dehydrogen- (64) Francis, K.; Gadda, G. Probing the Chemical Steps of
ase Reaction. J. Am. Chem. Soc. 1979, 101 (13), 3634−3640. Nitroalkane Oxidation Catalyzed by 2-Nitropropane Dioxygenase
(44) Cleland, W. W. Low-Barrier Hydrogen Bonds and Enzymatic with Solvent Viscosity, PH, and Substrate Kinetic Isotope Effects.
Catalysis. Arch. Biochem. Biophys. 2000, 382 (1), 1−5. Biochemistry 2006, 45 (46), 13889−13898.

213 https://doi.org/10.1021/acsbiomedchemau.4c00108
ACS Bio Med Chem Au 2025, 5, 204−214
ACS Bio & Med Chem Au pubs.acs.org/biomedchemau Article

(65) Feng, C.; Kedia, R. V.; Hazzard, J. T.; Hurley, J. K.; Tollin, G.;
Enemark, J. H. Effect of Solution Viscosity on Intramolecular Electron
Transfer in Sulfite Oxidase. Biochemistry 2002, 41 (18), 5816−5821.
(66) Vodovoz, M.; Gadda, G. Kinetic Solvent Viscosity Effects
Reveal a Protein Isomerization in the Reductive Half-Reaction of
Neurospora Crassa Class II Nitronate Monooxygenase. Arch. Biochem.
Biophys. 2020, 695, 108625.
(67) Muller, P. Glossary of Terms Used in Physical Organic
Chemistry (IUPAC Recommendations 1994). Pure Appl. Chem. 1994,
66 (5), 1077−1184.
(68) Calvert, J. G. Glossary of Atmospheric Chemistry Terms
(Recommendations 1990). Pure Appl. Chem. 1990, 62 (11), 2167−
2219.
(69) 'Base' in IUPAC Compendium of Chemical TerminologyInterna-
tional Union of Pure and Applied Chemistry; 3rd, Ed.; 2006. Online
version 3.0.1, 2019. https://doi.org/10.1351/goldbook.B00601

■ NOTE ADDED AFTER ASAP PUBLICATION

Due to a production error, the version of this paper that was
published ASAP on December 6, 2024, contained a typo-
graphical error in the definition of the SIE value in the second
paragraph of the “Effect of D2O Viscosity on the SIE of
PaD2HGDH Flavin Reduction with D-Malate” section. The
corrected version was reposted December 9, 2024.

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