FAD Prosthetic Group Research Articles

Discovery and biochemical characterization of thermostable glycerol oxidases

Alditol oxidases are promising tools for the biocatalytic oxidation of glycerol to more valuable chemicals. By integrating in silico bioprospecting with cell-free protein synthesis and activity screening, an effective pipeline was developed to rapidly identify enzymes that are active on glycerol. Three thermostable alditol oxidases from Actinobacteria Bacterium, Streptomyces thermoviolaceus, and Thermostaphylospora chromogena active on glycerol were discovered. The characterization of these three flavoenzymes demonstrated their glycerol oxidation activities, preference for alkaline conditions, and excellent thermostabilities with melting temperatures higher than 75 °C. Structural elucidation of the alditol oxidase from Actinobacteria Bacterium highlighted a constellation of side chains that engage the substrate through several hydrogen bonds, a histidine residue covalently bound to the FAD prosthetic group, and a tunnel leading to the active site. Upon computational simulations of substrate binding, a double mutant targeting a residue pair at the tunnel entrance was created and found to display an improved thermal stability and catalytic efficiency for glycerol oxidation. The hereby described alditol oxidases form a valuable panel of oxidative biocatalysts that can perform regioselective oxidation of glycerol and other polyols.Key points• Rapid pipeline designed to identify putative oxidases• Biochemical and structural characterization of alditol oxidases• Glycerol oxidation to more valuable derivativesGraphical

The Role of the si-Face Tyrosine of a Homodimeric Ferredoxin-NADP+ Oxidoreductase from Bacillus subtilis during Complex Formation and Redox Equivalent Transfer with NADP+/H and Ferredoxin.

In the crystal structure of ferredoxin-NADP+ oxidoreductase from Bacillus subtilis (BsFNR), Tyr50 stacks on the si-face of the isoalloxazine ring portion of the FAD prosthetic group. This configuration is highly conserved among the homodimeric ferredoxin-NAD(P)+ oxidoreductases (FNR) from Gram-positive bacteria and photosynthetic bacteria. In this report, pre-steady state reactions of Tyr50 variants with NADP+/NADPH and ferredoxin from B. subtilis (BsFd) were examined with stopped-flow spectrophotometry to assess the effects of the mutation on the formation of FNR-substrate complexes and following redox equivalent transfer. Mixing oxidized BsFNRs with NADPH resulted in a rapid complex formation followed by a rate-limiting hydride transfer. The substitution substantially modulated the intensity of the charge transfer absorption band and decreased the observed hydride transfer rates compared to the wild type. Reduction of the Y50W mutant by NADPH proceeded in a monophasic manner, while the Y50G and Y50S mutants did in biphasic phases. The reduced Tyr50 mutants hardly promoted the reduction of NADP+. Mixing oxidized BsFNRs with reduced BsFd resulted in the reduction of the FNRs. The observed FNR reduction rates of the three variants were comparable, but in the Y50G and Y50S mutants, the amount of the reduced FNR at the rapid phase was decreased, and a slow FNR reduction phase was observed. The obtained results suggest that the replacements of Tyr50 with Gly and Ser permitted the conformational change in the reduced form, which induced an asymmetric kinetic behavior between the protomers of the homodimeric BsFNR.

Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase.

Clinically relevant disease-causing variants of the human dihydrolipoamide dehydrogenase (hLADH, hE3), a common component of the mitochondrial α-keto acid dehydrogenase complexes, were characterized using a multipronged approach to unravel the molecular pathomechanisms that underlie hLADH deficiency. The G101del and M326V substitutions both reduced the protein stability and triggered the disassembly of the functional/obligate hLADH homodimer and significant FAD losses, which altogether eventually manifested in a virtually undetectable catalytic activity in both cases. The I12T-hLADH variant proved also to be quite unstable, but managed to retain the dimeric enzyme form; the LADH activity, both in the forward and reverse catalytic directions and the affinity for the prosthetic group FAD were both significantly compromised. None of the above three variants lent themselves to an in-depth structural analysis via X-ray crystallography due to inherent protein instability. Crystal structures at 2.89 and 2.44 Å resolutions were determined for the I318T- and I358T-hLADH variants, respectively; structure analysis revealed minor conformational perturbations, which correlated well with the residual LADH activities, in both cases. For the dimer interface variants G426E-, I445M-, and R447G-hLADH, enzyme activities and FAD loss were determined and compared against the previously published structural data.

Evolutionary and structural analyses of the NADPH oxidase family in eukaryotes reveal an initial calcium dependency

Reactive oxygen species are unstable molecules generated by the partial reduction of dioxygen. NADPH oxidases are a ubiquitous family of enzymes devoted to ROS production. They fuel an array of physiological roles in different species and are chemically demanding enzymes requiring FAD, NADPH and heme prosthetic groups in addition to either calcium or a various number of cytosolic mediators for activity. These activating partners are exclusive components that partition and distinguish the NOX members from one another. To gain insight into the evolution of these activating mechanisms, and in general in their evolutionary history, we conducted an in-depth phylogenetic analysis of the NADPH oxidase family in eukaryotes. We show that all characterized NOXs share a common ancestor, which comprised a fully formed catalytic unit. Regarding the activation mode, we identified calcium-dependency as the earliest form of NOX regulation. The protein-protein mode of regulation would have evolved more recently by gene-duplication with the concomitant loss of the EF-hands motif region. These more recent events generated the diversely activated NOX systems as observed in extant animals and fungi.

Kinetic and structural insight into a role of the re-face Tyr328 residue of the homodimer type ferredoxin-NADP+ oxidoreductase from Rhodopseudomonas palustris in the reaction with NADP+/NADPH

Among the thioredoxin reductase-type ferredoxin-NAD(P)+ oxidoreductase (FNR) family, FNR from photosynthetic purple non‑sulfur bacterium Rhodopseudomonas palustris (RpFNR) is distinctive because the predicted residue on the re-face of the isoalloxazine ring portion of the FAD prosthetic group is a tyrosine. Here, we report the crystal structure of wild type RpFNR and kinetic analyses of the reaction of wild type, and Y328F, Y328H and Y328S mutants with NADP+/NADPH using steady state and pre-steady state kinetic approaches.The obtained crystal structure of wild type RpFNR confirmed the presence of Tyr328 on the re-face of the isoalloxazine ring of the FAD prosthetic group through the unique hydrogen bonding of its hydroxyl group. In the steady state assays, the substitution results in the decrease of Kd for NADP+ and KM for NADPH in the diaphorase assay; however, the kcat values also decreased significantly. In the stopped-flow spectrophotometry, mixing oxidized RpFNRs with NADPH and reduced RpFNRs with NADP+ resulted in rapid charge transfer complex formation followed by hydride transfer. The observed rate constants for the hydride transfer in both directions were comparable (>400 s−1). The substitution did not drastically affect the rate of hydride transfer, but substantially slowed down the subsequent release and re-association of NADP+/NADPH in both directions. The obtained results suggest that Tyr328 stabilizes the stacking of C-terminal residues on the isoalloxazine ring portion of the FAD prosthetic group, which impedes the access of NADP+/NADPH on the isoalloxazine ring portions, in turn, enhancing the release of the NADP+/NADPH and/or reaction with electron transfer proteins.

Role of the Re-Face Tyr Residue Stacked on the FAD Prosthetic Group in Ferredoxin-Nadp+ Oxidoreductase from Rhodopseudomonas palustris during Catalysis with NADP+/H

Role of the Re-Face Tyr Residue Stacked on the FAD Prosthetic Group in Ferredoxin-Nadp+ Oxidoreductase from Rhodopseudomonas palustris during Catalysis with NADP+/H

Stilbene epoxidation and detoxification in a Photorhabdus luminescens-nematode symbiosis

Members of the gammaproteobacterial Photorhabdus genus share mutualistic relationships with Heterorhabditis nematodes, and the pairs infect a wide swath of insect larvae. Photorhabdus species produce a family of stilbenes, with two major components being 3,5-dihydroxy-4-isopropyl-trans-stilbene (compound 1) and its stilbene epoxide (compound 2). This family of molecules harbors antimicrobial and immunosuppressive activities, and its pathway is responsible for producing a nematode "food signal" involved in nematode development. However, stilbene epoxidation biosynthesis and its biological roles remain unknown. Here, we identified an orphan protein (Plu2236) from Photorhabdus luminescens that catalyzes stilbene epoxidation. Structural, mutational, and biochemical analyses confirmed the enzyme adopts a fold common to FAD-dependent monooxygenases, contains a tightly bound FAD prosthetic group, and is required for the stereoselective epoxidation of compounds 1 and 2. The epoxidase gene was dispensable in a nematode-infective juvenile recovery assay, indicating the oxidized compound is not required for the food signal. The epoxide exhibited reduced cytotoxicity toward its producer, suggesting this may be a natural route for intracellular detoxification. In an insect infection model, we also observed two stilbene-derived metabolites that were dependent on the epoxidase. NMR, computational, and chemical degradation studies established their structures as new stilbene-l-proline conjugates, prolbenes A (compound 3) and B (compound 4). The prolbenes lacked immunosuppressive and antimicrobial activities compared with their stilbene substrates, suggesting a metabolite attenuation mechanism in the animal model. Collectively, our studies provide a structural view for stereoselective stilbene epoxidation and functionalization in an invertebrate animal infection model and provide new insights into stilbene cellular detoxification.

Directionality of the Reversible Reduction/Oxidation Reactions Catalyzed by Ferredoxin-NAD(P)H Oxidoreductases from Phototrophic and Heterotrophic Bacteria

Ferredoxin-NAD(P)+ oxidoreductases (FNRs) of photoautotroph Chlorobaculum tepidum (CtFNR) and heterotroph Bacillus subtilis (BsFNR) are homo-dimeric flavoprotein exhibiting a significant conservation of structural topology with bacterial NADPH-thioredoxin reductase. Although the redox reaction catalyzed by FNR is theoretically reversible, under the physiological conditions the direction of the reaction is assumed to be optimized toward either NADP+ reduction in photosynthetic process or NADPH oxidation in non-photosynthetic one. In this presentation the directionality of the redox reactions with NADP+/H and the structure-function relationships will be discussed based on the pre-steady-state kinetics. NADPH oxidation and NADP+ reduction reactions by BsFNR occurred with rapid formations of charge transfer complexes (CTCs) followed by the rate-limiting hydride transfer. The rate of the NADPH oxidation direction in the hydride transfer step was much faster than that of NADP+ reduction direction (500 s−1 vs. < 10 s−1), indicating NADPH oxidation was favored in BsFNR-catalyzing reaction. The deletions of the C-terminal amino acid residues almost did not affect the directionality. NADPH oxidation by CtFNR yielded a rapid formation of CTCs followedby the rate-limiting hydride transfer. The amount of reduced CtFNR at the equilibrium was in part even in the presence of ∼50-fold excess NADPH. NADP+ reduction by CtFNR provided no charge transfer band and the observed rate for the NADP+ reduction by CtFNR was rather slow (∼ 5 s−1), suggesting the formation of CTC was the rate-limiting. The hydride transfer rates in both directions were estimated to be comparable, indicating redox reaction with NADP+/H was reversible in the case of CtFNR. The depletion of the C-terminal residues turns out CtFNR to be NADPH oxidizing enzyme, suggesting that the C-terminal residues are required for tuning the redox properties of the FAD prosthetic group.

Replacement of Tyr50 stacked on the si-face of the isoalloxazine ring of the flavin adenine dinucleotide prosthetic group modulates Bacillus subtilis ferredoxin-NADP(+) oxidoreductase activity toward NADPH.

Ferredoxin-NAD(P)(+) oxidoreductases ([EC 1.18.1.2], [EC 1.18.1.3], FNRs) from green sulfur bacteria, purple non-sulfur bacteria and most of Firmicutes, such as Bacillus subtilis (BsFNR) are homo-dimeric flavoproteins homologous to bacterial NADPH-thioredoxin reductase. These FNRs contain two unique aromatic residues stacked on the si- and re-face of the isoalloxazine ring moiety of the FAD prosthetic group whose configurations are often found among other types of flavoproteins including plant-type FNR and flavodoxin, but not in bacterial NADPH-thioredoxin reductase. To investigate the role of the si-face Tyr50 residue in BsFNR, we replaced Tyr50 with Gly, Ser, and Trp and examined its spectroscopic properties and enzymatic activities in the presence of NADPH and ferredoxin (Fd) from B. subtilis (BsFd). The replacement of Tyr50 to Gly (Y50G), Ser (Y50S), and Trp (Y50W) in BsFNR resulted in a blue shift of the FAD transition bands. The Y50G and Y50S mutations enhanced the FAD fluorescence emission, whereas those of the wild type and Y50W mutant were quenched. All three mutants decreased thermal stabilities compared to wild type. Using a diaphorase assay, the k cat values for the Y50G and Y50S mutants in the presence of NADPH and ferricyanide were decreased to less than 5% of the wild type activity. The Y50W mutant retained approximately 20% reactivity in the diaphorase assay and BsFd-dependent cytochrome c reduction assay relative to wild type. The present results suggest that Tyr50 modulates the electronic properties and positioning of the prosthetic group.

Empirical valence bond simulations of the hydride transfer step in the monoamine oxidase B catalyzed metabolism of dopamine

Monoamine oxidases (MAOs) A and B are flavoenzymes responsible for the metabolism of biogenic amines such as dopamine, serotonin and noradrenaline. In this work, we present a comprehensive study of the rate-limiting step of dopamine degradation by MAO B, which consists in the hydride transfer from the methylene group of the substrate to the flavin moiety of the FAD prosthetic group. This article builds on our previous quantum chemical study of the same reaction using a cluster model (Vianello et al., Eur J Org Chem 2012; 7057), but now considering the full dimensionality of the hydrated enzyme with extensive configurational sampling. We show that MAO B is specifically tuned to catalyze the hydride transfer step from the substrate to the flavin moiety of the FAD prosthetic group and that it lowers the activation barrier by 12.3 kcal mol⁻¹ compared to the same reaction in aqueous solution, a rate enhancement of more than nine orders of magnitude. Taking into account the deprotonation of the substrate prior to the hydride transfer reaction, the activation barrier in the enzyme is calculated to be 16.1 kcal mol⁻¹, in excellent agreement with the experimental value of 16.5 kcal mol⁻¹. Additionally, we demonstrate that the protonation state of the active site residue Lys296 does not have an influence on the hydride transfer reaction.

Structure Analysis and Comparative Characterization of the Cytochrome c′ and Flavocytochrome c from Thermophilic Purple Photosynthetic Bacterium Thermochromatium tepidum

The thermodynamic and spectroscopic properties of two soluble electron transport proteins, cytochrome (Cyt) c' and flavocytochrome c, isolated from thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum were examined and compared with those of the corresponding proteins from a closely related mesophilic bacterium Allochromatium (Alc.) vinosum. These proteins share sequence identities of 82% for the cytochromes c' and 86% for the flavocytochromes c. Crystal structures of the two proteins have been determined at high resolutions. Differential scanning calorimetry and denaturing experiments show that both proteins from Tch. tepidum are thermally and structurally much more stable than their mesophilic counterparts. The denaturation temperature of Tch. tepidum Cyt c' was 22 °C higher than that of Alc. vinosum Cyt c', and the midpoints of denaturation using guanidine hydrochloride were 2.0 and 1.2 M for the Tch. tepidum and Alc. vinosum flavocytochromes c, respectively. The enhanced stabilities can be interpreted on the basis of the structural and sequence information obtained in this study: increased number of hydrogen bonds formed between main chain nitrogen and oxygen atoms, more compact structures and reduced number of glycine residues. Many residues with large side chains in Alc. vinosum Cyt c' are substituted by alanines in Tch. tepidum Cyt c'. Both proteins from Tch. tepidum exhibit high structural similarities to their counterparts from Alc. vinosum, and the different residues between the corresponding proteins are mainly located on the surface and exposed to the solvent. Water molecules are found in the heme vicinity of Tch. tepidum Cyt c' and form hydrogen bonds with the heme ligand and C-terminal charged residues. Similar bound waters are also found in the vicinity of one heme group in the diheme subunit of Tch. tepidum flavocytochrome c. Electron density map of the Tch. tepidum flavocytochrome c clearly revealed the presence of disulfur atoms positioned between two cysteine residues at the active site near the FAD prosthetic group. The result strongly suggests that flavocytochrome c is involved in the sulfide oxidation in vivo. Detailed discussion is given on the relationships between the crystal structures and the spectroscopic properties observed for these proteins.

Mechanistic and Stereochemical Studies of Glycine Oxidase from Bacillus subtilis Strain R5

Glycine oxidase gene from a strain of Bacillus subtilis was cloned and expressed in Escherichia coli. The purified enzyme was found, by mass spectrometry, to have a protein M(r) of 40763 (value of 40761.6 predicted from DNA sequence) and a FAD prosthetic group M(r) of 785.1 (theoretical value of 785.5). Glycine oxidase optimally catalyzes the conversion of glycine and oxygen into glyoxylate, hydrogen peroxide, and ammonia. Using samples of [2-RS-(3)H(2),2-(14)C]-, [2-R-(3)H,2-(14)C]-, and [2-S-(3)H,2-(14)C]glycine, we found that in the overall process H(Si) is removed. Incubation of the enzyme with [2-RS-(3)H(2),2-(14)C]glycine under anaerobic conditions, when only the reducing half of the reaction can occur, led to the recovery of 98.5% of the original glycine, which had the same (3)H:(14)C ratio as the starting substrate. The primary isotope effect was studied using [2-(2)H(2)]glycine, and we found that the specificity constants, k(cat)/K(M), for the protio and deuterio substrates were 1.46 x 10(3) and 1.05 x 10(2) M(-1) s(-1), respectively. Two alternative mechanisms for FAD-containing oxidases that involve either the intermediacy of a FADH(2)-imino acid complex or an amino acid covalently linked to FAD, formed via a carbanion, have been considered. The current knowledge of the mechanisms is reviewed, and we argue that a mechanism involving the FADH(2)-imino acid complex can be dissected to satisfactorily explain some of puzzling observations for which the carbanion mechanism was originally conceived. Furthermore, our results, together with observations in the literature, suggest that the interaction of glycine with the enzyme occurs within a tight ternary complex, which is protected from the protons of the medium.

Pirlindole analogs as probes of the active site of Monoamine Oxidase A

Pirlindole analogs are antidepressants that selectively inhibit mitochondrial monoamine oxidase A. New analogs were synthesized to probe the interaction with the FAD prosthetic group. Previous work (Hynson, R.M.G., Wouters, J. and Ramsay, R.R. (2003) Biochem. Pharmacol. 65, 1867–74) showed that changing the position of the pirlindole nitrogen relative to the N5 of the isoalloxazine ring altered the intensity of the spectral shift at 500 nm. New derivatives masking the pirlindole nitrogen or changing it to C or O provide experimental support for the position of the inhibitor docked in the active site.Supported by INTAS 99‐00433

Inherited Glutathione Reductase Deficiency and Plasmodium falciparum Malaria—A Case Study

In Plasmodium falciparum-infected red blood cells (RBCs), the flavoenzyme glutathione reductase (GR) regenerates reduced glutathione, which is essential for antioxidant defense. GR utilizes NADPH produced in the pentose phosphate shunt by glucose-6-phosphate dehydrogenase (G6PD). Thus, conditions affecting host G6PD or GR induce increased sensitivity to oxidants. Hereditary G6PD deficiency is frequent in malaria endemic areas and provides protection against severe malaria. Furthermore, GR deficiency resulting from insufficient saturation of the enzyme with its prosthetic group FAD is common. Based on these naturally occurring phenomena, GR of malaria parasites and their host cells represent attractive antimalarial drug targets. Recently we were given the opportunity to examine invasion, growth, and drug sensitivity of three P. falciparum strains (3D7, K1, and Palo Alto) in the RBCs from three homozygous individuals with total GR deficiency resulting from mutations in the apoprotein. Invasion or growth in the GR-deficient RBCs was not impaired for any of the parasite strains tested. Drug sensitivity to chloroquine, artemisinin, and methylene blue was comparable to parasites grown in GR-sufficient RBCs and sensitivity towards paraquat and sodium nitroprusside was only slightly enhanced. In contrast, membrane deposition of hemichromes as well as the opsonizing complement C3b fragments and phagocytosis were strongly increased in ring-infected RBCs of the GR-deficient individuals compared to ring-infected normal RBCs. Also, in one of the individuals, membrane-bound autologous IgGs were significantly enhanced. Thus, based on our in vitro data, GR deficiency and drug-induced GR inhibition may protect from malaria by inducing enhanced ring stage phagocytosis rather than by impairing parasite growth directly.

The development of biochemistry and molecular biology in China

The development of biochemistry and molecular biology in China

Characterization of a Naturally Occurring Mutation (Ile-12 to Thr) Close to Prosthetic Group FAD in Human Dihydrolipoamide Dehydrogenase

It catalyzes the reoxidation of the dihydrolipoyl prosthetic group attached to the lysyl residue(s) of the acyltransferase components of the three α-keto acid dehydrogenase complexes and to the hydrogen-carrier protein of the glycine cleavage system. It is a homodimeric flavoenzyme containing one FAD at each subunit.

Crystal Structures of NADH:FMN Oxidoreductase (EmoB) at Different Stages of Catalysis

EDTA has become a major organic pollutant in the environment because of its extreme usage and resistance to biodegradation. Recently, two critical enzymes, EDTA monooxygenase (EmoA) and NADH:FMN oxidoreductase (EmoB), belonging to the newly established two-component flavin-diffusible monooxygenase family, were identified in the EDTA degradation pathway in Mesorhizobium sp. BNC1. EmoA is an FMNH2-dependent enzyme that requires EmoB to provide FMNH2 for the conversion of EDTA to ethylenediaminediacetate. To understand the molecular basis of this FMN-mediated reaction, the crystal structures of the apo-form, FMN.FMN complex, and FMN.NADH complex of EmoB were determined at 2.5 angstroms resolution. The structure of EmoB is a homotetramer consisting of four alpha/beta-single-domain monomers of five parallel beta-strands flanked by five alpha-helices, which is quite different from those of other known two-component flavin-diffusible monooxygenase family members, such as PheA2 and HpaC, in terms of both tertiary and quaternary structures. For the first time, the crystal structures of both the FMN.FMN and FMN.NADH complexes of an NADH:FMN oxidoreductase were determined. Two stacked isoalloxazine rings and nicotinamide/isoalloxazine rings were at a proper distance for hydride transfer. The structures indicated a ping-pong reaction mechanism, which was confirmed by activity assays. Thus, the structural data offer detailed mechanistic information for hydride transfer between NADH to an enzyme-bound FMN and between the bound FMNH2 and a diffusible FMN.

Role in anaerobiosis of the isoenzymes for Saccharomyces cerevisiae fumarate reductase encoded by OSM1 and FRDS1

Saccharomyces cerevisiae possesses both a cytoplasmic and a mitochondrial fumarate reductase, encoded by FRDS1 and OSM1, respectively. While previous studies have shown that mutants lacking FRDS1 and OSM1 cannot grow under anaerobiosis (Arikawa et al., 1998), the physiological role of fumarate reductase (FR) remains poorly understood. Here, we report that an osm1 frds1 mutant is unable to grow anaerobically, even with glutamate as a sole nitrogen source, when succinate can be produced by the TCA oxidative branch. We also show that the growth of the mutant is not restored by adding acetoin, an alternative sink for NADH oxidation, but it is at least partly restored by the addition of oxygen or menadione, which can oxidize FADH(2) in addition to NADH. These data indicate that the growth inhibition of the mutant is due to an inability to reoxidize FAD, rather than an indirect effect on NADH or an inability to produce succinate per se. During anaerobic growth, FRDS1 expression was two to eight times higher than that of OSM1, and fumarate reductase activity was higher in the osm1 mutant than in the frds1 mutant. FRDS1 expression was induced by anaerobiosis, and this induction was abolished in a rox1 mutant. We conclude that the formation of succinate is strictly required for the reoxidation of FADH(2) during anaerobiosis, and that it is regulated through the control of FRDS1 expression when oxygen is limiting. Based on these data, we discuss the potential role of fumarate reductase in the regeneration of the FAD-prosthetic group of essential flavoproteins.

Erv2p: Characterization of the Redox Behavior of a Yeast Sulfhydryl Oxidase

The FAD prosthetic group of the ERV/ALR family of sulfhydryl oxidases is housed at the mouth of a 4-helix bundle and communicates with a pair of juxtaposed cysteine residues that form the proximal redox active disulfide. Most of these enzymes have one or more additional distal disulfide redox centers that facilitate the transfer of reducing equivalents from the dithiol substrates of these oxidases to the isoalloxazine ring where the reaction with molecular oxygen occurs. The present study examines yeast Erv2p and compares the redox behavior of this ER luminal protein with the augmenter of liver regeneration, a sulfhydryl oxidase of the mitochondrial intermembrane space, and a larger protein containing the ERV/ALR domain, quiescin-sulfhydryl oxidase (QSOX). Dithionite and photochemical reductions of Erv2p show full reduction of the flavin cofactor after the addition of 4 electrons with a midpoint potential of -200 mV at pH 7.5. A charge-transfer complex between a proximal thiolate and the oxidized flavin is not observed in Erv2p consistent with a distribution of reducing equivalents over the flavin and distal disulfide redox centers. Upon coordination with Zn2+, full reduction of Erv2p requires 6 electrons. Zn2+ also strongly inhibits Erv2p when assayed using tris(2-carboxyethyl)phosphine (TCEP) as the reducing substrate of the oxidase. In contrast to QSOX, Erv2p shows a comparatively low turnover with a range of small thiol substrates, with reduced Escherichia coli thioredoxin and with unfolded proteins. Rapid reaction studies confirm that reduction of the flavin center of Erv2p is rate-limiting during turnover with molecular oxygen. This comparison of the redox properties between members of the ERV/ALR family of sulfhydryl oxidases provides insights into their likely roles in oxidative protein folding.

Mechanism of action of a flavin-containing monooxygenase

Elimination of nonnutritional and insoluble compounds is a critical task for any living organism. Flavin-containing monooxygenases (FMOs) attach an oxygen atom to the insoluble nucleophilic compounds to increase solubility and thereby increase excretion. Here we analyze the functional mechanism of FMO from Schizosaccharomyces pombe using the crystal structures of the wild type and protein-cofactor and protein-substrate complexes. The structure of the wild-type FMO revealed that the prosthetic group FAD is an integral part of the protein. FMO needs NADPH as a cofactor in addition to the prosthetic group for its catalytic activity. Structures of the protein-cofactor and protein-substrate complexes provide insights into mechanism of action. We propose that FMOs exist in the cell as a complex with a reduced form of the prosthetic group and NADPH cofactor, readying them to act on substrates. The 4alpha-hydroperoxyflavin form of the prosthetic group represents a transient intermediate of the monooxygenation process. The oxygenated and reduced forms of the prosthetic group help stabilize interactions with cofactor and substrate alternately to permit continuous enzyme turnover.