The kinetics of reoxidation of reduced benzylamine oxidase.

1. The mechanism of reoxidation of reduced benzylamine oxidase has been investigated at different pH between 6 and 10 by steady-state and transient-state kinetic methods. 2. The reoxidation process involves minimally a second-order interaction between reduced enzyme and oxygen leading to the formation of a spectrally modified enzyme intermediate, and a subsequent first-order step converting this intermediate into free enzyme. The variation with pH of rate constants according to such a reaction scheme is reported. 3. Under aerobic conditions the oxygen-independent reaction represents the main rate-limiting step in the catalytic process at alkaline pH. At neutral or acid pH the interaction between reduced enzyme and oxygen becomes mainly rate-limiting, indicating that the concentration of oxygen may be a critical factor controlling enzyme activity under physiological conditions. 4. The spectrally modified intermediate formed during the reoxidation process exhibits a difference-absorption band centered around 290 nm in comparison to free enzyme, and an additional difference-absorption band at 470 nm in comparison to reduced enzyme. These data indicate that formation of the intermediate, besides leading to a reappearance of the 470-nm absorption band disappearing on reduction of the enzyme, results in a spectral perturbation of one or several aromatic amino-acid residues in the protein. This perturbation could possibly reflect a conformational change of the enzymes.     

Kinetic isotope effects on the catalytic activity of pig-plasma benzylamine oxidase.

1. Isotope effects on the catalytic activity of benzylamine oxidase at pH 7 and 9 have been studied by steady-state and transient-state kinetics methods, using [alpha,alpha-2H]benzylamine as the substrate. 2. Replacement of the alpha-hydrogen atoms in benzylamine by deuterium has no significant effect on substrate-binding to benzylamine oxidase, neither does it affect the rate of reoxidation of the reduced form of the enzyme. Conversion of the primarily formed enzyme-substrate complex into the reduced enzyme species, however, exhibits an isotope effect of about 3. 3. The data obtained are consistent with a mechanism in which reduction of benzylamine oxidase takes place by a rapid pre-equilibration between enzyme and substrate to form an amine-pyridoxal Schiff-base, which is then tautomerized by a comparatively slow prototropic shift to an amino aldehyde-pyridoxamine Schiff-base from which there is a rapid hydrolytic release of the aldehyde product corresponding to the amine substrate. Proton abstraction from the alpha-carbon of the amine moiety in the primary Schiff-base appears to be at least partially rate-limiting for the tautomerization step, and hence for the entire process of enzyme reduction.     

The kinetic mechanism of beef kidney D-aspartate oxidase

The mechanism of action of the flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been investigated by steady-state and stopped flow kinetic studies using D-aspartate and O2 as substrates in 50 mM KPi, 0.3 mM EDTA, pH 7.4, 4 degrees C. Steady-state results indicate that a ternary complex containing enzyme, O2, and substrate (or product) is an obligatory intermediate in catalysis. The kinetic parameters are turnover number = 11.1 s-1, Km(D-Asp) = 2.2 x 10(-3) M, Km(O2) = 1.7 x 10(-4) M. Rapid reaction studies show that 1) the reductive half reaction is essentially irreversible with a maximum rate of reduction of 180 s-1; 2) the free reduced enzyme cannot be the species which is reoxidized during turnover since its reoxidation by oxygen (second order rate constant equal to 5.3 x 10(2) M-1 s-1) is too slow to be of relevance in catalysis; 3) reduced enzyme can bind a ligand rapidly and be reoxidized as a complex at a rate faster than that observed for the free reduced enzyme; 4) the rate of reoxidation of reduced enzyme by oxygen during turnover is dependent on both O2 and D-aspartate concentrations (second order rate constant of reaction between O2 and reduced enzyme-substrate complex equal to 6.2 x 10(4) M-1 s-1); and 5) the rate-limiting step in catalysis occurs after reoxidation of the enzyme and before its reduction in the following turnover. A mechanism involving reduction of enzyme by substrate, dissociation of product from reduced enzyme, binding of a second molecule of substrate to the reduced enzyme, and reoxidation of the reduced enzyme-substrate complex is proposed for the enzyme-catalyzed oxidation of D-aspartate.     

Stopped-flow spectrophotometric characterization of enzymic reaction intermediates in the anaerobic reduction of pig-plasma benzylamine oxidase by amine substrates.

Reduction of benzylamine oxidase by p-methoxybenzylamine under anaerobic conditions leads to biphasic absorbance changes at 470 nm. These reflect the intermediate formation of an enzyme substrate complex with spectral properties different from those of native enzyme and fully reduced enzyme. The spectrally modified enzyme-substrate complex exhibits a broad difference absorption band centered around 360 nm. The transient accumulation of this intermediate during reaction can be conveniently followed by stopped-flow techniques at wavelengths between 320 and 360 nm, where contributions from the subsequent reduction of the enzymic 470-nm chromophore are of minor significance. 2. Analogous intermediates exhibiting similar absorption spectra seem to be formed on reduction of the enzyme by benzylamine and other amine substrates which were tested. Substitution of benzylamine as the reducing substrate by [alpha, alpha-2H]benzylamine results in a decreased accumulation of the spectrally modified intermediate. This indicates that its formation is preceded by deprotonation of the alpha-carbon of the amine substrate. 3. Circular dichroism spectra of benzylamine oxidase exhibit a positive band at 360 nm, lending support to the previous conclusion that benzylamine oxidase is a pyridoxal enzyme. Formation of the spectrally modified enzyme-substrate complex then most likely reflects the prototropic shift converting an amine-pyridoxal Schiff-base obtained by rapid pre-equilibration between enzyme and substrate into an aldehyde-pyridoxamine Schiff-base.     

Properties of copper-free pig kidney amine oxidase: role of topa quinone

Copper removal from pig kidney amine oxidase containing Cu/topaquinone (TPQ) has been obtained using CN(-) in the presence of the poor substrate p-(dimethylamino)benzylamine. Upon removal of copper, the enzyme loses its activity while the TPQ cofactor remains in its oxidized form. The addition of copper to the apo-form fully restores the active enzyme. The CN(-) treatment in the presence of sodium dithionite or good substrates (cadaverine or benzylamine) also removes copper but the TPQ cofactor is irreversibly reduced and the addition of copper does not regenerate the active enzyme. Ni(II) and Zn(II) do not bind the apo-protein in contrast to Co(II) which is incorporated to the same extent as Cu(II). However, Co-reconstituted enzyme only shows a very low activity. These results demonstrate that copper is essential for the catalytic mechanism because it maintains the correct active site geometry.     

Spectroscopic and catalytic properties of Rhus vernicifera laccase depleted in type 2 copper.

1. The type 2 copper in Rhus vernicifera laccase was completely removed without loss of other types of copper. The properties of this protein derivative and the role of type 2 copper in the catalytic action of laccase was investigated. 2. The molar extinction coefficient at 614 nm of the blue chromophore decreases from 5700 to 4700 cm-1 on removal of type 2 copper. There are no apparent absorption changes at other wavelengths in the visible or near ultraviolet region when this copper is taken away. The electron-paramagnetic-resonance (epr) parameter A parallel and the linewidth of type 1 Cu2+ decreases on removal of type 2 copper. 3. The rate of reduction of type 1 Cu2+ is not affected by removal of type 2 copper but the reduction of the two-electron acceptor is greatly impaired. These results strongly support the idea that type 1 Cu2+ is the primary site for electron transfer between substrate and enzyme and that the two-electron acceptor in the native enzyme is reduced by simultaneous electron transfer from reduced types 1 and 2 copper. 4. Reoxidation of types 1 and 3 copper and the formation of the oxygen intermediate are the same processes in native and type-2-depleted enzyme. These observations suggests that type 2 copper is not involved in the formation and rapid decay of the oxygen intermediate and that it is not necessary for the stabilization of this intermediate. 5. Two new epr signals are observed on reoxidation of reduced type-2-depleted laccase. One is temporarily formed on re-reduction of reoxidized enzyme and it is suggested that it might arise from copper, possibly type 3 copper. The other one is stable for hours and it is proposed that it might come from a modified oxygen intermediate.     

The binding of carbon monoxide to cytochrome c oxidase.

The effect of CO on the optical absorbance spectrum of partially reduced cytochrome c oxidase has been studied. The changes at 432 and 590 nm suggest that the cytochrome alpha2/3+ - CO compound is formed preferentially and that concomitantly a second electron is taken up by the enzyme. From the CO-induced changes at 830 nm it is concluded that in the partially reduced enzyme addition of CO causes reoxidation of the copper component of cytochrome c oxidase. Addition of CO to partially reduced enzyme (2 electrons per 4 metal ions) also brings about a decrease in the intensities of electron paramagnetic resonance signals of high-spin heme iron near g = 6 and of the low-spin heme at g = 2.6. Concomitantly both the low-spin heme a signal at g = 3 and the copper signal at g = 2 increase in intensity. These results demonstrate that formation of the reduced diamagnetic cytochrome a3 - CO compound is accompanied by reoxidation of both the copper component detectable by electron paramagnetic resonance and possibly also by cytochrome a.     

Lentil seedlings amine oxidase: preparation and properties of the copper-free enzyme

The reaction of copper-free lentil seedlings amine oxidase with substrates has been studied. While devoid of catalytic activity, this enzyme preparation is still able to oxidize two moles of substrate and to release two moles of aldehyde and two moles of ammonia per mole of dimeric protein. The same stoichiometry has been determined on the native enzyme in the absence of oxygen. Although copper is essential for the reoxidation of the reduced enzyme, a binding of oxygen to the copper-free protein has been demonstrated.     

Changes in the copper centres of benzylamine oxidase from pig plasma during the catalytic cycle.

The Cu²⁺ sites in benzylamine oxidase for various points in the catalytic cycle have been studied by 35GHz EPR spectroscopy in conjunction with the rapid freeze technique. No evidence has been obtained for reduction or oxidation of Cu²⁺ in any intermediate. The only change detected is in the E reduced intermediate for which the ligand environment of one of the two Cu²⁺ sites is modified. This observation provides direct evidence for the participation of Cu²⁺ in the catalytic mechanism and is consistent with other reports that the enzyme has only one active site.     

Pyrroloquinoline quinone as cofactor in galactose oxidase (EC 1.1.3.9)

Galactose oxidase from Dactylium dendroides was shown to contain one molecule of covalently bound pyrroloquinoline quinone (PQQ/enzyme molecule. From the spectroscopic characteristics reported for the enzyme forms, a mechanistic role for PQQ could be deduced. In analogy with other quinoproteins, the initial formation of a PQQ-substrate adduct is proposed. Following internal hydrogen transfer, leading to aldehyde product and reduced pyrroloquinoline quinone, reoxidation of the organic cofactor with molecular oxygen could be mediated by the PQQ-liganded copper ion with concomitant formation of hydrogen peroxide. With PQQ as an additional (two-electron) redox center the occurrence of a "superoxidized" enzyme form must be considered. Possible consequences of this view, in relation to a physiological function of the enzyme and interpretation of ESR data, are discussed.     

Differences between the reactivities of two pyridine nucleotides in the rapid reduction process and the reoxidation process of adrenodoxin reductase.

The reaction process of adrenodoxin reductase with NADPH and NADH were investigated. The appearance of new intermediate with a broad absorption band at around 520 nm has been detected by rapid-scan stopped-flow spectrophotometry. Although the formation of this intermediate is more rapid with NADPH than with NADH, the rates of the subsequent decay to the fully reduced state are almost identical (Kobs values were 20.5 and 16.0s-1). These results indicate that the new intermediate is the complex formed between the oxidized enzyme and reduced pyridine nucleotide (enzyme-substrate complex), and that subsequent decay of the intermidiate is caused by a two-electron transfer process from the reduced pyridine nucleotide to the enzyme flavin. On the other hand, spectral and kinetic properties in the steady state of the reoxidation reaction of the enzyme reduced with NADPH and NADH were somewhat different. The rate of reoxidation of the enzyme under aerobic conditions from the reduced state to the oxidized state was 6.5 times faster when a 10-fold molar excess of NADH was used than when NADPH of the same concentration was used. This result is consistent with the fact that the NADH-dependent oxidase activity was 6.4 times greater than that dependent on NADPH. During reoxidation of the reduced enzyme under aerobic conditions in the presence of an excess of NADPH or NADH, the EPR spectra indicated the formation of the flavin semiquinone radical species. Similarly, the formation of semiquinone was observed in the absorption spectrum with either NADPH or NADH under the same conditions as in the EPR measurement. The intensity of the semiquinone signal on EPR was considerably smaller with NADH than with NADPH. These results suggest that NADP+ complex with the enzyme semiquinone protects the radical from oxidation by oxygen to a greater extent than NAD+, and consequently the semiquinone is easier to detect with NADPH than with NADH.     

Isotopic probes yield microscopic constants: separation of binding energy from catalytic efficiency in the bovine plasma amine oxidase reaction

Bovine plasma amine oxidase catalyzes the oxidative deamination of primary amines. The reaction can be viewed as two half-reactions: enzyme reduction by substrate followed by enzyme reoxidation by dioxygen. Anaerobic stopped-flow kinetic measurements of the first half-reaction indicate very large deuterium isotope effects for benzylamine, m-tyramine, and dopamine, Dk = 13.5 +/- 1.3, which are ascribed to an intrinsic isotope effect. From the insensitivity of these isotope effects to amine concentration, stopped-flow data provide substrate dissociation constants, K1, and rate constants for the C-H bond cleavage step, k3, directly. Steady-state isotope effects have also been measured for benzylamine and six ring-substituted phenethylamines. Whereas a small range of values for kcat, 0.38-1.2 s-1, and Dkcat, 5.4-8.8, is observed, kcat/Km = 1.3 X 10(2) to 3.8 X 10(4) M-1 S-1 and D(kcat/Km) = 5.6-16.1 indicate a marked effect of ring substituent. As described earlier [Miller, S., & Klinman, J.P. (1982) Methods Enzymol. 87, 711], the availability of an intrinsic isotope effect for an enzymatic reaction permits calculation of microscopic constants from steady-state data. By employment of a minimal mechanism for bovine plasma amine oxidase involving a single precatalytic and multiple postcatalytic enzyme-substrate complexes, equations have been derived that allow calculation of k3 and K1 when DKeq congruent to 1 less than Dk. Unexpectedly, in the case of K1, we have shown that this parameter can be calculated from steady-state parameters without the requirement for an intrinsic isotope effect. This result should have general application to both ping-pong and sequential ternary-complex enzyme mechanisms. Of significance for future applications of steady-state isotope effects to the calculation of microscopic constants, values for K1 and k3 derived from steady-state parameters and single turnover measurements indicate excellent agreement. Compilation of parameters among six ring-substituted phenethylamines reveals alteration in delta G for enzyme-substrate complex formation by 2.8 kcal/mol, together with an essentially invariant rate constant for C-H bond activation. A detailed discussion of the relevance of these findings to the interrelationship of binding energy and catalytic efficiency in enzyme reactions is presented.     

Stopped-flow studies on the mechanism of oxidation of N-methyl-4-phenyltetrahydropyridine by bovine liver monoamine oxidase B

The kinetic mechanism of monoamine oxidase B involves either a binary or a ternary complex, depending on the substrate. In this study, stopped-flow kinetic data provide direct evidence for ternary complexes not only of reduced enzyme, oxygen, and product but also of reduced enzyme, oxygen, and substrate, both for benzylamine and for the tertiary amine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, the mechanism for a given substrate is not exclusive but, rather, is determined by competition between the alternate pathways as a result of different rate constants for the oxidation of the reduced enzyme, the reduced enzyme-product complex, and the reduced enzyme-substrate complex, as well as the different dissociation constants for the complexes. Comparison of the rate constants obtained from the stopped-flow studies with steady-state data indicates that the overall rate of reaction for the oxidation of MPTP by monoamine oxidase is dominated by the reductive step, but for benzylamine the steady-state rate is determined by a complex function of the rates of both the reductive and oxidative half-reactions.     

Mechanism of the electron transfer catalyst DsbB from Escherichia coli

The membrane protein DsbB from Escherichia coli is essential for disulfide bond formation and catalyses the oxidation of the periplasmic dithiol oxidase DsbA by ubiquinone. DsbB contains two catalytic disulfide bonds, Cys41-Cys44 and Cys104-Cys130. We show that DsbB directly oxidizes one molar equivalent of DsbA in the absence of ubiquinone via disulfide exchange with the 104-130 disulfide bond, with a rate constant of 2.7 x 10 M(-1) x s(-1). This reaction occurs although the 104-130 disulfide is less oxidizing than the catalytic disulfide bond of DsbA (E(o)' = -186 and -122 mV, respectively). This is because the 41-44 disulfide, which is only accessible to ubiquinone but not to DsbA, is the most oxidizing disulfide bond in a protein described so far, with a redox potential of -69 mV. Rapid intramolecular disulfide exchange in partially reduced DsbB converts the enzyme into a state in which Cys41 and Cys44 are reduced and thus accessible for reoxidation by ubiquinone. This demonstrates that the high catalytic efficiency of DsbB results from the extreme intrinsic oxidative force of the enzyme.     

Kinetic mechanism of monoamine oxidase A

Steady-state kinetic data for monoamine oxidase A in crude extracts suggest an exclusively ping-pong mechanism, in contrast to those for monoamine oxidase B, which indicate alternate mechanisms involving either a binary or ternary complex. In this study, with use of purified monoamine oxidase A, steady-state data for the inhibition by D-amphetamine of the oxidation of primary amines indicate the possibility of a ternary complex mechanism for monoamine oxidase A also. Stopped-flow studies demonstrate that the rate of reoxidation of reduced enzyme is enhanced by substrates but not by the product, 1-methyl-4-phenylpyridinium. Thus, for the A enzyme, the ternary complex with substrate, but not product, is reoxidized at a faster rate than the free, reduced enzyme. For both the A and B forms of monoamine oxidase, the mechanism is determined by competition between alternate pathways on the basis of the relative rate constants and dissociation constants.     

Phenylalanine hydroxylase from Chromobacterium violaceum is a copper-containing monooxygenase. Kinetics of the reductive activation of the enzyme

Pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum contains a stoichiometric amount of copper (Cu2+, 1 mol/mol of enzyme). Electron paramagnetic resonance spectroscopy of the enzyme indicates that it is a type II copper-containing protein. The oxidized enzyme must be reduced by a single electron to be catalytically active. Dithiothreitol was found to be an effective reducing agent for the enzyme. Electron paramagnetic resonance data and kinetic results indicate the formation of an enzyme-thiol complex during the aerobic reduction of the enzyme by dithiothreitol. 6,7-Dimethyltetrahydropterin also reductively activates the enzyme, but only in the presence of the substrate, and is kinetically less effective than dithiothreitol. The metal center is not reoxidized as a result of normal turnover. However, the data indicate an alternative pathway exists that results in slow reoxidation of the enzyme. The 4a-hydrate of 6-methyltetrahydropterin (4a-carbinolamine) is observed during turnover of the enzyme. This intermediate is also observed during the reaction catalyzed by the iron-containing mammalian enzyme, suggesting that the mechanism of oxygen activation is similar for both enzymes.     

Loss of enzyme activity during turnover of the Bacillus cereus β-lactamase catalysed hydrolysis of β-lactams due to loss of zinc ion

Metallo-beta-lactamases are zinc-ion-dependent and are known to exist either as mononuclear or as dinuclear enzymes. The kinetics and mechanism of hydrolysis of the native zinc Bacillus cereus metallo-beta-lactamase (BcII) have been investigated under pre-steady-state conditions at different pHs and zinc-ion concentrations. Biphasic kinetics are observed for the hydrolysis of cefuroxime and benzylpenicillin with submicromolar concentrations of enzyme and zinc. The initial burst of product formation far exceeds the concentration of enzyme and the subsequent slower rate of hydrolysis is attributed to a branched kinetic pathway. The pH and metal-ion dependence of the microscopic rate constants of this branching were determined, from which it is concluded that two enzyme species with different metal-to-enzyme stoichiometries are formed during catalytic turnover. The dizinc enzyme is responsible for the fast route but during the catalytic cycle it slowly loses the less tightly bound zinc ion via the branching route to give an inactive monozinc enzyme; the latter is only catalytic following the uptake of a second zinc ion. The rate constant for product formation from the dinuclear enzyme and the branching rate constant show a sigmoidal dependence on pH indicative of important ionizing groups with pK (a)s of 9.0 +/- 0.1 and 8.2 +/- 0.1, respectively. The rate constant for the regeneration of enzyme activity depends on zinc-ion concentration. This unusual behaviour is attributed to an intrinsic property of metallo hydrolytic enzymes that depend on a metal bound water both as a ligand for the second metal ion and as the nucleophile which is consumed during hydrolysis of the substrate and so has to be replaced to maintain the catalytic cycle.     

An important lysine residue in copper/quinone-containing amine oxidases

The interaction of xenon with copper/6-hydroxydopa (2,4,5-trihydroxyphenethylamine) quinone (TPQ) amine oxidases from the plant pulses lentil (Lens esculenta) and pea (Pisum sativum) (seedlings), the perennial Mediterranean shrub Euphorbia characias (latex), and the mammals cattle (serum) and pigs (kidney), were investigated by NMR and optical spectroscopy of the aqueous solutions of the enzymes. (129)Xe chemical shift provided evidence of xenon binding to one or more cavities of all these enzymes, and optical spectroscopy showed that under 10 atm of xenon gas, and in the absence of a substrate, the plant enzyme cofactor (TPQ), is converted into its reduced semiquinolamine radical. The kinetic parameters of the analyzed plant amine oxidases showed that the k(c) value of the xenon-treated enzymes was reduced by 40%. Moreover, whereas the measured K(m) value for oxygen and for the aromatic monoamine benzylamine was shown to be unchanged, the K(m) value for the diamine putrescine increased remarkably after the addition of xenon. Under the same experimental conditions, the TPQ of bovine serum amine oxidase maintained its oxidized form, whereas in pig kidney, the reduced aminoquinol species was formed without the radical species. Moreover the k(c) value of the xenon-treated pig enzyme in the presence of both benzylamine and cadaverine was shown to be dramatically reduced. It is proposed that the lysine residue at the active site of amine oxidase could be involved both in the formation of the reduced TPQ and in controlling catalytic activity.     

The action of plasma amine oxidase on beta-haloamines. Evidence for proton abstraction in the oxidative reaction.

The action of plasma amine oxidase upon beta-Br-ethylamine beta-Cl-ethylamine, beta-OH-phenylethylamine, and beta-Cl-phenylethylamine was examined. Beta-Br-ethylamine is a substrate and irreversible inactivator of the enzyme. The enzyme becomes covalently labeled by the inactivator. Approximately 2 mol of inactivator are incorporated per mol of enzyme (MW 170,000). The reduced enzyme is not inactivated. The enzyme catalyzes the elimination of HCl from beta-Cl-phenylethylamine to produce phenylacetaldehyde. The rate of the elimination reaction is comparable to the normal oxidative reaction. We conclude that the occurrence of this elimination reaction establishes the ability of the enzyme to catalyze proton abstraction from C-1 of the substrate and that proton abstraction occurs during the catalytic oxidation normally catalyzed by plasma amine oxidase. Beta-Cl-ethylamine is only oxidized to corresponding aldehyde. Beta-OH-phenylethylamine is neither oxidized, nor does elimination occur. It is a competitive inhibitor in the oxidation of benzylamine and in the elimination of HCl from beta-Cl-phenylethylamine.     

Spectroscopic studies on the photoreaction of choline oxidase, a flavoprotein, with covalently bound flavin

Formation of the anionic flavosemiquinone was observed spectrophotometrically during the anaerobic photo-irradiation of Alcaligenes sp. choline oxidase in the presence of EDTA. Further irradiation slowly converted the semiquinone form into the fully reduced state. The presence of a catalytic amount of riboflavin greatly enhances the photoreduction rate not only to the semiquinone state but also to the fully reduced state. This semiquinone species has low reactivity toward the substrate, choline or betaine aldehyde, as well as toward oxygen. This low reactivity toward oxygen is unique to the semiquinone form of a flavoprotein oxidase. The oxidized enzyme forms a complex with betaine, the product of the enzymatic reaction of choline oxidase. The dissociation constant for this complex was found to be 17 mM by spectroscopic titration. Anaerobic photo-irradiation of the enzyme with a saturating amount of betaine in the absence of EDTA produces, with no detectable semiquinone formation, an absorption spectrum which resembles (but significantly differs from) that of the fully reduced form. This species was found to comprise two flavin species. One of them is rapidly oxidized to the oxidized form by oxygen and is thus assigned as the fully reduced state. The other is converted slowly to the oxidized form upon aerobic standing in the dark. We tentatively assigned this latter species as a C(4a)-adduct. Formaldehyde was detected as a product of this photoreaction. The amount of formaldehyde formed coincided with that of the fully reduced enzyme. On the basis of the results obtained we propose a mechanism of the photoreaction of the enzyme in the presence of betaine where a C(4a)-adduct and the fully reduced enzyme via an N(5)-adduct are formed. Betaine also affects the dithionite reduction. In the dithionite reduction of the oxidized enzyme, the semiquinone species is an intermediate in the conversion of the oxidized to the fully reduced form, while the reduction of the oxidized enzyme-betaine complex with dithionite produces the fully reduced form without any significant formation of the semiquinone species.