P-hydroxybenzoate Hydroxylase Research Articles

Role of Acinetobacter baylyi Crc in Catabolite Repression of Enzymes for Aromatic Compound Catabolism

Here, we describe for the first time the Crc (catabolite repression control) protein from the soil bacterium Acinetobacter baylyi. Expression of A. baylyi crc varied according to the growth conditions. A strain with a disrupted crc gene showed the same growth as the wild type on a number of carbon sources. Carbon catabolite repression by acetate and succinate of protocatechuate 3,4-dioxygenase, the key enzyme of protocatechuate breakdown, was strongly reduced in the crc strain, whereas in the wild-type strain it underwent strong catabolite repression. This strong effect was not based on transcriptional regulation because the transcription pattern of the pca-qui operon (encoding protocatechuate 3,4-dioxygenase) did not reflect the derepression in the absence of Crc. pca-qui transcript abundance was slightly increased in the crc strain. Lack of Crc dramatically increased the mRNA stability of the pca-qui transcript (up to 14-fold), whereas two other transcripts (pobA and catA) remained unaffected. p-Hydroxybenzoate hydroxylase activity, encoded by pobA, was not significantly different in the absence of Crc, as protocatechuate 3,4-dioxygenase was. It is proposed that A. baylyi Crc is involved in the determination of the transcript stability of the pca-qui operon and thereby effects catabolite repression.

1P-016 計算的手法を用いた酵素設計 : 部位特異的変異によるパラ水酸化安息香酸水酸化酵素の基質特異性の改変(蛋白質-構造,第47回日本生物物理学会年会)

1P-016 計算的手法を用いた酵素設計 : 部位特異的変異によるパラ水酸化安息香酸水酸化酵素の基質特異性の改変(蛋白質-構造,第47回日本生物物理学会年会)

The FAD Cofactor of RebC Shifts to an IN Conformation upon Flavin Reduction,

RebC is a putative flavin hydroxylase functioning together with RebP to carry out a key step in the biosynthesis of rebeccamycin. To probe the mechanism of flavin-based chemistry in RebC, we solved the structure of RebC with reduced flavin. Upon flavin reduction, the RebC crystal undergoes a change in its unit cell dimension concurrent with a 5 Å movement of the isoalloxazine ring, positioning the flavin ring adjacent to the substrate-binding pocket. Additionally, a disordered helix becomes ordered upon flavin reduction, closing off one side of the substrate-binding pocket. This structure, along with previously reported structures, increases our understanding of the RebC enzyme mechanism, indicating that either the reduction of the flavin itself or binding of substrate is sufficient to drive major conformational changes in RebC to generate a closed active site. Our finding that flavin reduction seals the active site such that substrate cannot enter suggests that our reduced flavin RebC structure is off-pathway and that substrate binding is likely to precede flavin reduction during catalysis. Along with kinetic data presented here, these structures suggest that the first cycle of catalysis in RebC may resemble that of p-hydroxybenzoate hydroxylase, with substrate binding promoting flavin reduction.

Crystal Structure of the Pyocyanin Biosynthetic Protein PhzS

The human pathogen Pseudomonas aeruginosa produces pyocyanin, a blue-pigmented phenazine derivative, which is known to play a role in virulence. Pyocyanin is produced from chorismic acid via the phenazine pathway, nine proteins encoded by a gene cluster. Phenazine-1-carboxylic acid, the initial phenazine formed, is converted to pyocyanin in two steps that are catalyzed by the enzymes PhzM and PhzS. PhzM is an adenosylmethionine dependent methyltransferase, and PhzS is a flavin dependent hydroxylase. It has been shown that PhzM is only active in the physical presence of PhzS, suggesting that a protein-protein interaction is involved in pyocyanin formation. Such a complex would prevent the release of 5-methyl-phenazine-1-carboxylate, the putative intermediate, and an apparently unstable compound. Here, we describe the three-dimensional structure of PhzS, solved by single anomalous dispersion, at a resolution of 2.4 A. The structure reveals that PhzS is a member of the family of aromatic hydroxylases characterized by p-hydroxybenzoate hydroxylase. The flavin cofactor of PhzS is in the solvent exposed out orientation typically seen in unliganded aromatic hydroxylases. The PhzS flavin, however, appears to be held in a strained conformation by a combination of stacking interactions and hydrogen bonds. The structure suggests that access to the active site is gained via a tunnel on the opposite side of the protein from where the flavin is exposed. The C-terminal 23 residues are disordered as no electron density is present for these atoms. The probable location of the C-terminus, near the substrate access tunnel, suggests that it may be involved in substrate binding as has been shown for another structural homologue, RebC. This region also may be an element of a PhzM-PhzS interface. Aromatic hydroxylases have been shown to catalyze electrophilic substitution reactions on activated substrates. The putative PhzS substrate, however, is electron deficient and unlikely to act as a nucleophile, suggesting that PhzS may use a different mechanism than its structural relatives.

Mechanism and Regulation of the Two-component FMN-dependent Monooxygenase ActVA-ActVB from Streptomyces coelicolor

The ActVA-ActVB system from Streptomyces coelicolor is a two-component flavin-dependent monooxygenase involved in the antibiotic actinorhodin biosynthesis. ActVB is a NADH:flavin oxidoreductase that provides a reduced FMN to ActVA, the monooxygenase that catalyzes the hydroxylation of dihydrokalafungin, the precursor of actinorhodin. In this work, using stopped-flow spectrophotometry, we investigated the mechanism of hydroxylation of dihydrokalafungin catalyzed by ActVA and that of the reduced FMN transfer from ActVB to ActVA. Our results show that the hydroxylation mechanism proceeds with the participation of two different reaction intermediates in ActVA active site. First, a C(4a)-FMN-hydroperoxide species is formed after binding of reduced FMN to the monooxygenase and reaction with O(2). This intermediate hydroxylates the substrate and is transformed to a second reaction intermediate, a C(4a)-FMN-hydroxy species. In addition, we demonstrate that reduced FMN can be transferred efficiently from the reductase to the monooxygenase without involving any protein.protein complexes. The rate of transfer of reduced FMN from ActVB to ActVA was found to be controlled by the release of NAD(+) from ActVB and was strongly affected by NAD(+) concentration, with an IC(50) of 40 microm. This control of reduced FMN transfer by NAD(+) was associated with the formation of a strong charge.transfer complex between NAD(+) and reduced FMN in the active site of ActVB. These results suggest that, in Streptomyces coelicolor, the reductase component ActVB can act as a regulatory component of the monooxygenase activity by controlling the transfer of reduced FMN to the monooxygenase.

Monomer Formation and Function of p‐Hydroxybenzoate Hydroxylase in Reverse Micelles and in Dimethylsulfoxide/Water Mixtures

It has previously been postulated that the dimeric form of the flavoprotein p-hydroxybenzoate hydroxylase (PHBH) is important for catalysis. Here it is demonstrated that the monomeric form of PHBH is active. In a water/AOT/isooctane reverse micellar system, the function of the monomeric and dimeric forms of PHBH could be observed separately by varying the size of the micelles. A considerable decrease in the K(M) value for p-hydroxybenzoate (POHB) was found for monomeric PHBH, accompanied by a 1.5-fold decrease in enzymatic activity. The same tendency was observed when monomers of PHBH were formed by adding DMSO to the buffer. The FAD in PHBH and PHBH labeled with the fluorescence dye Alexa488 was investigated by time-resolved fluorescence anisotropy to observe monomer formation in water/DMSO mixtures. Monomer formation of PHBH occurred gradually with increasing DMSO content in the mixture. Pure PHBH monomers were detected at DMSO concentrations of 30 % (v/v) and higher.

Molecular cloning and functional characterization of  the genes encoding benzoate and p-hydroxybenzoate degradation by the halophilic Chromohalobacter sp. strain HS-2

Chromohalobacter sp. strain HS-2 was isolated from salted fermented clams and analyzed for the ability to grow on benzoate and p-hydroxybenzoate as the sole carbon and energy source. HS-2 was characterized as moderately halophilic, with an optimal NaCl concentration of 10%. The genes encoding the benzoate metabolism were cloned into a cosmid vector, sequenced, and then analyzed to reveal the benzoate (benABCD) and catechol (catBCA) catabolic genes, both of which are flanked on either side by LysR-type transcriptional regulator (catR) and membrane transport protein for benzoate (benE) in the gene order catRBCAbenABCDE. Near the large cat-ben cluster, a p-hydroxybenzoate hydroxylase gene (pobA) and two putative regulatory genes (pcaQ and pobR) were additionally detected. The HS-2 genes involved in benzoate and p-hydroxybenzoate degradation are tightly clustered within a c. 19 kb region, and show quite a different genetic organization from those of other benzoate catabolic genes. Reverse transcriptase-PCR experiments show that benzoate induces the expression of benzoate 1,2-dioxygenase, catechol 1,2-dioxygenase, and protocatechuate 3,4-dioxygenase while p-hydroxybenzoate only induced the expression of p-hydroxybenzoate hydroxylase. When expressed in Escherichia coli, benzoate 1,2-dioxygenase (BenABC) and p-hydroxybenzoate hydroxylase (PobA) transformed benzoate and p-hydroxybenzoate into cis-benzoate dihydrodiol and protocatechuate, respectively.

Toward accurate barriers for enzymatic reactions: QM/MM case study on p-hydroxybenzoate hydroxylase

The hydroxylation reaction catalyzed by p-hydroxybenzoate hydroxylase has been investigated by quantum mechanical/molecular mechanical (QM/MM) calculations at different levels of QM theory. The solvated enzyme was modeled (approximately 23,000 atoms in total, 49 QM atoms). The geometries of reactant and transition state were optimized for ten representative pathways using semiempirical (AM1) and density functional (B3LYP) methods as QM components. Single-point calculations at B3LYP/MM optimized geometries were performed with local correlation methods [LMP2, LCCSD(T0)] and augmented triple-zeta basis sets. A careful validation of the latter approach with regard to all computational parameters indicates convergence of the QM contribution to the computed barriers to within approximately 1 kcal mol(-1). Comparison with the available experimental data supports this assessment.

Expression, purification, crystallization and initial crystallographic characterization of thep-hydroxybenzoate hydroxylase fromCorynebacterium glutamicum

p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction and plays an important role in the biodegradation of aromatic compounds. PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH(2)PO(4) and K(2)HPO(4) as precipitants. X-ray diffraction data were collected to a maximum resolution of 2.5 A on a synchrotron beamline. The crystal belongs to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 94.72, c = 359.68 A, gamma = 120 degrees . The asymmetric unit contains two molecules, corresponding to a packing density of 2.65 A(3) Da(-1). The structure was solved by molecular replacement. Structure refinement is in progress.

Bioproduction of p-hydroxybenzoate from renewable feedstock by solvent-tolerant Pseudomonas putida S12

Pseudomonas putida strain S12palB1 was constructed that produces p-hydroxybenzoate from renewable carbon sources via the central metabolite l-tyrosine. P. putida S12palB1 was based on the platform strain P. putida S12TPL3, which has an optimised carbon flux towards l-tyrosine. Phenylalanine ammonia lyase (Pal) was introduced for the conversion of l-tyrosine into p-coumarate, which is further converted into p-hydroxybenzoate by endogenous enzymes. p-Hydroxybenzoate hydroxylase (PobA) was inactivated to prevent the degradation of p-hydroxybenzoate. These modifications resulted in stable accumulation of p-hydroxybenzoate at a yield of 11% (C-mol C-mol −1) on glucose or on glycerol in shake flask cultures. In a glycerol-limited fed-batch fermentation, a final p-hydroxybenzoate concentration of 12.9 mM (1.8 g l −1) was obtained, at a yield of 8.5% (C-mol C-mol −1). A 2-fold increase of the specific p-hydroxybenzoate production rate ( q p) was observed when l-tyrosine was supplied to a steady-state C-limited chemostat culture of P. putida S12palB1. This implied that l-tyrosine availability was the bottleneck for p-hydroxybenzoate production under these conditions. When p-coumarate was added instead, q p increased by a factor 4.7, indicating that Pal activity is the limiting factor when sufficient l-tyrosine is available. Thus, two major leads for further improvement of the p-hydroxybenzoate production by P. putida S12palB1 were identified.

Peptide mass fingerprinting- and 2-DE/MS-based analysis of the biodegradation potential for monocyclic aromatic hydrocarbons in Pseudomonas sp.

The combined analysis of peptide mass fingerprinting and 2-DE/MS using the induced and selected protein spots following growth of Pseudomonas sp. DU102 on benzoate or p-hydroxybenzoate revealed not only alpha- and beta-subunits of protocatechuate 3,4-dioxygenase but also catechol 1,2-dioxygenase responsible for ortho-pathway through ring-cleavage of aromatic compounds. Toluate 1,2-dioxygenase and p-hydroxybenzoate hydroxylase were also identified. Purification of intradiol dioxygenases such as catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase from the benzoate or p-hydroxybenzoate culture makes it possible to trace the biodegradation pathway of strain DU102 for monocyclic aromatic hydrocarbons. Interestingly, vanillin-induced protocatechuate 3,4-dioxygenase was identical in amino acid sequences with protocatechuate 3,4-dioxygenase from p-hydroxybenzoate.

A Systematic and Comprehensive Combinatorial Approach to Simultaneously Improve the Activity, Reaction Specificity, and Thermal Stability of p-Hydroxybenzoate Hydroxylase

We have simultaneously improved the activity, reaction specificity, and thermal stability of p-hydroxybenzoate hydroxylase by means of systematic and comprehensive combinatorial mutagenesis starting from available single mutations. Introduction of random mutations at the positions of four cysteine and eight methionine residues provided 216 single mutants as stably expressed forms in Escherichia coli host cells. Four characteristics, hydroxylase activity toward p-hydroxybenzoate (main activity), protocatechuate-dependent NADPH oxidase activity (sub-activity), ratio of sub-activity to main activity (reaction specificity), and thermal stability, of the purified mutants were determined. To improve the above characteristics for diagnostic use of the enzyme, 11 single mutations (C152V, C211I, C332A, M52V, M52Q, M110L, M110I, M213G, M213L, M276Q, and M349A) were selected for further combinatorial mutagenesis. All possible combinations of the mutations provided 18 variants with double mutations and further combinatorial mutagenesis provided 6 variants with triple mutations and 9 variants with quadruple mutations with the simultaneously improved four properties.

Development of an l-glutamate biosensor using the coimmobilization of l-glutamate dehydrogenase and p-hydroxybenzoate hydroxylase on a Clark-type electrode

Abstract A bienzyme-based Clark electrode was developed for the interference-free determination of l -glutamate. This sensor is based on the specific dehydrogenation by l -glutamate dehydrogenase (GLDH, EC 1.4.1.3) in combination with p-hydroxybenzoate hydroxylase (HBH, EC 1.14.13.2). The enzymes were entrapped by a poly(carbamoyl) sulfonate hydrogel on a Teflon membrane. The principle of the determination scheme is as follows: the specific detecting enzyme, GLDH, catalyses the specific dehydrogenation of l -glutamate consuming NADP+. The product, NADPH, initiates the irreversible hydroxylation of p-hydroxybenzoate by HBH in the presence of oxygen. This results in a detectable signal due to the HBH-enzymatic consumptions of dissolved oxygen in the measurement of l -glutamate. The sensor has a fast steady-state measuring time of 20 s with a quick response (2 s) and a short recovery (1 min). It shows a linear detection range between 10 μM and 1.5 mM l -glutamate and a detection limit of 5 μM. A Teflon membrane, which is used to fabricate the sensor, makes the determination to avoid interferences from different amino acids and electroactive substances.

Time-resolved fluorescence analysis of the mobile flavin cofactor in p-hydroxybenzoate hydroxylase

Conformational heterogeneity of the FAD cofactor in p-hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations; the 'in' conformation with the isoalloxazine ring located in the active site, and the 'out' conformation with the isoalloxazine ring disposed towards the protein surface. Fluores- cence-lifetime analysis of these complexes revealed similar lifetime distributions for the 'in' and 'out' conformations. The reason for this is twofold. First, the active site of PHBH contains various potential fluorescence-quenching sites close to the flavin. Fluorescence analysis of uncomplexed PHBH Y222V and Y222A showed that Tyr222 is responsible for picosecond fluorescence quenching free enzyme. In addition, other potential quenching sites, including a tryptophan and two tyrosines involved in substrate binding, are located nearby. Since the shortest distance between these quenching sites and the isoalloxaz- ine ring differs only little on average, these aromatic residues are likely to contribute to fluorescence quenching. Second, the effect of flavin conformation on the fluorescence lifetime distribution is blurred by binding of the aromatic substrates: saturation with aromatic substrates induces highly efficient fluo- rescence quenching. The flavin conformation is therefore only reflected in the small relative contribu- tions of the longer lifetimes.

Development of a glucose-6-phosphate biosensor based on coimmobilized p-hydroxybenzoate hydroxylase and glucose-6-phosphate dehydrogenase

This work reports the development of an amperometric glucose-6-phosphate biosensor by coimmobilizing p-hydroxybenzoate hydroxylase (HBH) and glucose-6-phosphate dehydrogenase (G6PDH) on a screen-printed electrode. The principle of the determination scheme is as follows: G6PDH catalyzes the specific dehydrogenation of glucose-6-phosphate by consuming NADP +. The product, NADPH, initiates the irreversible the hydroxylation of p-hydroxybenzoate by HBH in the presence of oxygen to produce 3,4-dihydroxybenzoate, which results in a detectable signal due to its oxidation at the working electrode. The sensor shows a broad linear detection range between 2 μM and 1000 μM with a low detection limit of 1.2 μM. Also, it has a fast measuring time which can achieve 95% of the maximum current response in 20 s after the addition of a given concentration of glucose-6-phosphate with a short recovery time (2 min).

Real-time Enzyme Dynamics Illustrated with Fluorescence Spectroscopy of p-Hydroxybenzoate Hydroxylase

We have used the flavoenzyme p-hydroxybenzoate hydroxylase (PHBH) to illustrate that a strongly fluorescent donor label can communicate with the flavin via single-pair Förster resonance energy transfer (spFRET). The accessible Cys-116 of PHBH was labeled with two different fluorescent maleimides with full preservation of enzymatic activity. One of these labels shows overlap between its fluorescence spectrum and the absorption spectrum of the FAD prosthetic group in the oxidized state, while the other fluorescent probe does not have this spectral overlap. The spectral overlap strongly diminished when the flavin becomes reduced during catalysis. The donor fluorescence properties can then be used as a sensitive antenna for the flavin redox state. Time-resolved fluorescence experiments on ensembles of labeled PHBH molecules were carried out in the absence and presence of enzymatic turnover. Distinct changes in fluorescence decays of spFRET-active PHBH can be observed when the enzyme is performing catalysis using both substrates p-hydroxybenzoate and NADPH. Single-molecule fluorescence correlation spectroscopy on spFRET-active PHBH showed the presence of a relaxation process (relaxation time of 23 micros) that is related to catalysis. In addition, in both labeled PHBH preparations the number of enzyme molecules reversibly increased during enzymatic turnover indicating that the dimer-monomer equilibrium is affected.

QM/MM Free-Energy Perturbation Compared to Thermodynamic Integration and Umbrella Sampling: Application to an Enzymatic Reaction.

We used the free-energy perturbation (FEP) method in quantum mechanics/molecular mechanics (QM/MM) calculations to compute the free-energy profile of the hydroxylation reaction in the enzyme p-hydroxybenzoate hydroxylase (PHBH). k statistics were employed to analyze the FEP sampling including estimation of the sampling error. Various approximations of the free-energy perturbation method were tested. We find that it is adequate not only to freeze the density of the QM part during the dynamics at frozen QM geometry but also to approximate this density by electrostatic-potential-fitted point charges. It is advisable to include all atoms of a QM/MM link in the perturbation. The results of QM/MM-FEP for PHBH are in good agreement with those of thermodynamic integration and umbrella sampling.

Conformational Dynamics of the Isoalloxazine in Substrate-Free p-Hydroxybenzoate Hydroxylase: Single-Molecule Studies

p-Hydroxybenzoate hydroxylase (PHBH) is a homodimeric enzyme in which each subunit noncovalently binds one molecule of FAD in the active site. PHBH is a model system for how flavoenzymes regulate reactions with oxygen. We report single-molecule fluorescence studies of PHBH in the absence of substrate that provide data consistent with the hypothesis that a critical step in substrate binding is the movement of the isoalloxazine between an "in" conformation and a more exposed or "open" conformation. The isoalloxazine is observed to move between these conformations in the absence of substrate. Studies with the Y222A mutant form of PHBH suggest that the exposed conformation is fluorescent while the in-conformation is quenched. Finally, we note that many of the single-molecule-fluorescence trajectories reveal a conformational heterogeneity, with populations of the enzyme characterized by either fast or slow switching between the in- and open-conformations. Our data also allow us to hypothesize a model in which one flavin in the dimer inhibits the motion of the other.

Properties of p-Hydroxybenzoate Hydroxylase When Stabilized in Its Open Conformation

p-Hydroxybenzoate hydroxylase is extensively studied as a model for single-component flavoprotein monooxygenases. It catalyzes a reaction in two parts: (1) reduction of the FAD in the enzyme by NADPH in response to binding of p-hydroxybenzoate to the enzyme and (2) oxidation of reduced FAD with oxygen in an environment free from solvent to form a hydroperoxide, which then reacts with p-hydroxybenzoate to form an oxygenated product. These different reactions are coordinated through conformational rearrangements of the protein and the isoalloxazine ring during catalysis. Until recently, it has not been clear how p-hydroxybenzoate gains access to the buried active site. In 2002, a structure of a mutant form of the enzyme without substrate was published that showed an open conformation with solvent access to the active site [Wang, J., Ortiz-Maldonado, M., Entsch, B., Massey, V., Ballou, D., and Gatti, D. L. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 608-613]. The wild-type enzyme does not form high-resolution crystals without substrate. We hypothesized that the wild-type enzyme without substrate also forms an open conformation for binding p-hydroxybenzoate, but only transiently. To test this idea, we have studied the properties of two different mutant forms of the enzyme that are stabilized in the open conformation. These mutant enzymes bind p-hydroxybenzoate very fast, but with very low affinity, as expected from the open structure. The mutant enzymes are extremely inactive, but are capable of slowly forming small amounts of product by the normal catalytic pathway. The lack of activity results from the failure of the mutants to readily form the out conformation required for flavin reduction by NADPH. The mutants form a large fraction of an abnormal conformation of the reduced enzyme with p-hydroxybenzoate bound. This conformation of the enzyme is unreactive with oxygen. We conclude that transient formation of this open conformation is the mechanism for sequestering p-hydroxybenzoate to initiate catalysis. This overall study emphasizes the role that protein dynamics can play in enzymatic catalysis.

Kinetics of Proton-Linked Flavin Conformational Changes in p-Hydroxybenzoate Hydroxylase

p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction. Two structural features are coupled to control the reactivity of PHBH with NADPH: a proton-transfer network that allows protons to be passed between the sequestered active site and solvent and a flavin that adopts two positions: "in", where the flavin is near pOHB, and "out", where the flavin is near NADPH. PHBH uses the proton-transfer network to test for the presence of a suitable aromatic substrate before allowing the flavin to adopt the NADPH-accessible conformation. In this work, kinetic analysis of the His72Asn mutant, with a disrupted proton-transfer network, showed that flavin movement could occur in the presence or absence of NADPH but that NADPH stimulated movement to the reactive conformation required for hydride transfer. Substrate and solvent isotope effects on the transient kinetics of reduction of the His72Asn mutant showed that proton transfer was linked to flavin movement and that the conformational change occurred in a step separate from that of hydride transfer. Proton transfers during the reductive half-reaction were observed directly in the wild-type enzyme by performing experiments in the presence of a fluorescent pH-indicator dye in unbuffered solutions. NADPH binding caused rapid proton release from the enzyme, followed by proton uptake after flavin reduction. Solvent and substrate kinetic isotope effects showed that proton-coupled flavin movement and reduction also occurred in different steps in wild-type PHBH. These results allow a detailed kinetic scheme to be proposed for the reductive half-reaction of the wild-type enzyme. Three kinetic models considered for substrate-induced isomerization are analyzed in the Appendix.