Crystal structure of pyridoxine 4-oxidase from Mesorhizobium loti

Pyridoxine 4-oxidase (PNOX) from Mesorhizobium loti is a monomeric glucose–methanol–choline (GMC) oxidoreductase family enzyme, catalyzes FAD-dependent oxidation of pyridoxine (PN) into pyridoxal, and is the first enzyme in pathway I for the degradation of PN. The tertiary structures of PNOX with a C-terminal His₆-tag and PNOX–pyridoxamine (PM) complex were determined at 2.2 Å and at 2.1 Å resolutions, respectively. The overall structure consisted of FAD-binding and substrate-binding domains. In the active site, His460, His462, and Pro504 were located on the re-face of the isoalloxazine ring of FAD. PM binds to the active site through several hydrogen bonds. The side chains of His462 and His460 are located at 2.7 and 3.1 Å from the N4′ atom of PM. The activities of His460Ala and His462Ala mutant PNOXs were very low, and 460Ala/His462Ala double mutant PNOX exhibited no activity. His462 may act as a general base for the abstraction of a proton from the 4′-hydroxyl of PN. His460 may play a role in the binding and positioning of PN. The C4′ atom in PM is located at 3.2 Å, and the hydride ion from the C4′ atom may be transferred to the N5 atom of the isoalloxazine ring. The comparison of active site residues in GMC oxidoreductase shows that Pro504 in PNOX corresponds to Asn or His of the conserved His–Asn or His–His pair in other GMC oxidoreductases. The function of the novel proline residue was discussed.     

Crystal Structure of Sulfide:Quinone Oxidoreductase from Acidithiobacillus ferrooxidans: Insights into Sulfidotrophic Respiration and Detoxification

Sulfide:quinone oxidoreductase from the acidophilic and chemolithotrophic bacterium Acidithiobacillus ferrooxidans was expressed in Escherichia coli and crystallized, and its X-ray molecular structure was determined to 2.3 Å resolution for native unbound protein in space group P4₂2₁2 . The decylubiquinone-bound structure and the Cys160Ala variant structure were subsequently determined to 2.3 Å and 2.05 Å resolutions, respectively, in space group P6₂22  . The enzymatic reaction catalyzed by sulfide:quinone oxidoreductase includes the oxidation of sulfide compounds H₂S, HS⁻, and S²⁻ to soluble polysulfide chains or to elemental sulfur in the form of octasulfur rings; these oxidations are coupled to the reduction of ubiquinone or menaquinone. The enzyme comprises two tandem Rossmann fold domains and a flexible C-terminal domain encompassing two amphipathic helices that are thought to provide for membrane anchoring. The second amphipathic helix unwinds and changes its orientation in the hexagonal crystal form. The protein forms a dimer that could be inserted into the membrane to a depth of approximately 20 Å. It has an endogenous flavin adenine dinucleotide (FAD) cofactor that is noncovalently bound in the N-terminal domain. Several wide channels connect the FAD cofactor to the exterior of the protein molecule; some of the channels would provide access to the membrane. The ubiquinone molecule is bound in one of these channels; its benzoquinone ring is stacked between the aromatic rings of two conserved Phe residues, and it closely approaches the isoalloxazine moiety of the FAD cofactor. Two active-site cysteine residues situated on the re side of the FAD cofactor form a branched polysulfide bridge. Cys356 disulfide acts as a nucleophile that attacks the C4A atom of the FAD cofactor in electron transfer reaction. The third essential cysteine Cys128 is not modified in these structures; its role is likely confined to the release of the polysulfur product.     

13C, 15N, and 31P NMR studies on 6-hydroxy-L-nicotine oxidase from Arthrobacter oxidans

The interaction between the apoprotein of 6-hydroxy-L-nicotine oxidase from Arthrobacter oxidans and the prosthetic group FAD has been investigated by 13C, 15N, and 31P NMR techniques. The FAD prosthetic group was selectively enriched in 13C and 15N isotopes by adding isotopically labeled riboflavin derivatives to the growth medium of riboflavin-requiring mutant cells. In the oxidized state the chemical shift of the C(7) and C(8) atoms indicates that the xylene moiety of the isoalloxazine ring is embedded in a hydrophobic environment. The polarization of the isoalloxazine ring as a whole is, however, much more comparable to that of free flavin in a polar and protic environment than to free flavin in an apolar environment. The polarization of the ring system can be ascribed to strong hydrogen bonds between the apoprotein and the two carbonyl groups. The binding of the competitive inhibitor, 6-hydroxy-D-nicotine, influences the resonances of the C(4a) and the N(5) atoms strongly. It is suggested that these shifts are due to a strong hydrogen-bonding interaction between the N(5) atom and the inhibitor. On reduction all resonances, except those of the C(10a) and the N(1) atoms, shift upfield, indicating the increased electron density in the ring system. In the dithionite-reduced enzyme, the ring system is bent at the N(5) position. Due to the bending of the N(5) atom and the sp2 hybridized N(10) atom, electron density from the N(10) atom is reallocated at the C(4) carbonyl group. In contrast, in the substrate-reduced enzyme the N(5) atom is almost completely sp2 hybridized, yielding a rather planar isoalloxazine ring.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor

Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N⁶-(buta-2,3-dienyl)adenine (HA-8) and N⁶-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 Å resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.     

Asymmetric Dimeric Structure of Ferredoxin-NAD(P) Oxidoreductase from the Green Sulfur Bacterium Chlorobaculum tepidum: Implications for Binding Ferredoxin and NADP

Ferredoxin-NAD(P)⁺ oxidoreductase (FNR) catalyzes the reduction of NAD(P)⁺ to NAD(P)H with the reduced ferredoxin (Fd) during the final step of the photosynthetic electron transport chain. FNR from the green sulfur bacterium Chlorobaculum tepidum is functionally analogous to plant-type FNR but shares a structural homology to NADPH-dependent thioredoxin reductase (TrxR). Here, we report the crystal structure of C. tepidum FNR to 2.4 Å resolution, which reveals a unique structure-function relationship. C. tepidum FNR consists of two functional domains for binding FAD and NAD(P)H that form a homodimer in which the domains are arranged asymmetrically. One NAD(P)H domain is present as the open form, the other with the equivalent NAD(P)H domain as the relatively closed form. We used site-directed mutagenesis on the hinge region connecting the two domains in order to investigate the importance of the flexible hinge. The asymmetry of the NAD(P)H domain and the comparison with TrxR suggested that the hinge motion might be involved in pyridine nucleotide binding and binding of Fd. Surprisingly, the crystal structure revealed an additional C-terminal sub-domain that tethers one protomer and interacts with the other protomer by π-π stacking of Phe337 and the isoalloxazine ring of FAD. The position of this stacking Phe337 is almost identical with both of the conserved C-terminal Tyr residues of plant-type FNR and the active site dithiol of TrxR, implying a unique structural basis for enzymatic reaction of C. tepidum FNR.     

Structural Basis for Substrate Recognition and Specificity in Aklavinone-11-Hydroxylase from Rhodomycin Biosynthesis

In the biosynthesis of several anthracyclines, aromatic polyketides produced by many Streptomyces species, the aglycone core is modified by a specific flavin adenine dinucleotide (FAD)- and NAD(P)H-dependent aklavinone-11-hydroxylase. Here, we report the crystal structure of a ternary complex of this enzyme from Streptomyces purpurascens, RdmE, with FAD and the substrate aklavinone. The enzyme is built up of three domains, a FAD-binding domain, a domain involved in substrate binding, and a C-terminal thioredoxin-like domain of unknown function. RdmE exhibits structural similarity to aromatic hydroxylases from the p-hydroxybenzoate hydroxylase family, but unlike most other related enzymes, RdmE is a monomer. The substrate is bound in a hydrophobic pocket in the interior of the enzyme, and access to this pocket is provided through a different route than for the isoalloxazine ring of FAD—the backside of the ligand binding cleft. The architecture of the substrate binding pocket and the observed enzyme-aklavinone interactions provide a structural explanation for the specificity of the enzyme for non-glycosylated substrates with C9-R stereochemistry. The isoalloxazine ring of the flavin cofactor is bound in the “out” conformation but can be modeled in the “in” conformation without invoking large conformational changes of the enzyme. This model places the flavin ring in a position suitable for catalysis, almost perpendicular to the tetracyclic ring system of the substrate and with a distance of the C4a carbon atom of the isoalloxazine ring to the C-11 carbon atom of the substrate of 4.8 Å. The structure suggested that a Tyr224-Arg373 pair might be involved in proton abstraction at the C-6 hydroxyl group, thereby increasing the nucleophilicity of the aromatic ring system and facilitating electrophilic attack by the perhydroxy-flavin intermediate. Replacement of Tyr224 by phenylalanine results in inactive enzyme, whereas mutants at position Arg373 retain catalytic activity close to wild-type level. These data establish an essential role of residue Tyr224 in catalysis, possibly in aligning the substrate in a position suitable for catalysis.     

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 K_d for NADP⁺ and K_M for NADPH in the diaphorase assay; however, the k_cat 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.     

15N- and 13C-NMR investigations of glucose oxidase from Aspergillus niger

The apoprotein of glucose oxidase from Aspergillus niger was reconstituted with specifically 15N- and 13C-enriched FAD derivatives and investigated by 15N- and 13C-NMR spectroscopy. On the basis of the 15N-NMR results it is suggested that, in the oxidized state of glucose oxidase, hydrogen bonds are formed to the N(3) and N(5) positions of the isoalloxazine system. The hydrogen bond to N(3) is more pronounced than that to N(5) as compared with the respective hydrogen bonds formed between FMN and water. The resonance position of N(10) indicates a small decrease in sp2 hybridization compared to free flavin in water. Apparently the isoalloxazine ring is not planar at this position in glucose oxidase. Additional hydrogen bonds at the carbonyl groups of the oxidized enzyme-bound FAD were derived from the 13C-NMR results. A strong downfield shift observed for the C(4a) resonance may be ascribed in part to the decrease in sp2 hybridization at the N(10) position and to the polarization of the carbonyl groups at C(2) and C(4). The polarization of the isoalloxazine ring in glucose oxidase is more similar to FMN in water than to that of tetraacetyl-riboflavin in apolar solvents. In the reduced enzyme the N(1) position is anionic at pH 5.6. The pKa is shifted to lower pH values by at least 1 owing to the interaction of the FAD with the apoprotein. As in the oxidized state of the enzyme, a hydrogen bond is also formed at the N(3) position of the reduced flavin. The N(5) and N(10) resonances of the enzyme-bound reduced FAD indicate a decrease in the sp2 character of these atoms as compared with that of reduced FMN in aqueous solution. Some of the 15N- and 13C-resonance positions of the enzyme-bound reduced cofactor are markedly pH-dependent. The pH dependence of the N(5) and C(10a) resonances indicates a decrease in sp2 hybridization of the N(5) atom with increasing pH of the enzyme solution.     

Role of Thr(66) in porcine NADH-cytochrome b5 reductase in catalysis and control of the rate-limiting step in electron transfer

Site-directed mutagenesis of Thr(66) in porcine liver NADH-cytochrome b(5) reductase demonstrated that this residue modulates the semiquinone form of FAD and the rate-limiting step in the catalytic sequence of electron transfer. The absorption spectrum of the T66V mutant showed a typical neutral blue semiquinone intermediate during turnover in the electron transfer from NADH to ferricyanide but showed an anionic red semiquinone form during anaerobic photoreduction. The apparent k(cat) values of this mutant were approximately 10% of that of the wild type enzyme (WT). These data suggest that the T66V mutation stabilizes the neutral blue semiquinone and that the conversion of the neutral blue to the anionic red semiquinone form is the rate-limiting step. In the WT, the value of the rate constant of FAD reduction (k(red)) was consistent with the k(cat) values, and the oxidized enzyme-NADH complex was observed during the turnover with ferricyanide. This indicates that the reduction of FAD by NADH in the WT-NADH complex is the rate-limiting step. In the T66A mutant, the k(red) value was larger than the k(cat) values, but the k(red) value in the presence of NAD(+) was consistent with the k(cat) values. The spectral shape of this mutant observed during turnover was similar to that during the reduction with NADH in the presence of NAD(+). These data suggest that the oxidized T66A-NADH-NAD(+) ternary complex is a major intermediate in the turnover and that the release of NAD(+) from this complex is the rate-limiting step. These results substantiate the important role of Thr(66) in the one-electron transfer reaction catalyzed by this enzyme. On the basis of these data, we present a new kinetic scheme to explain the mechanism of electron transfer from NADH to one-electron acceptors including cytochrome b(5).     

Role of specific residues in coenzyme binding, charge-transfer complex formation, and catalysis in Anabaena ferredoxin NADP-reductase

Two transient charge-transfer complexes (CTC) form prior and upon hydride transfer (HT) in the reversible reaction of the FAD-dependent ferredoxin-NADP⁺ reductase (FNR) with NADP⁺/H, FNR_ox-NADPH (CTC-1), and FNR_rd-NADP⁺ (CTC-2). Spectral properties of both CTCs, as well as the corresponding interconversion HT rates, are here reported for several Anabaena FNR site-directed mutants. The need for an adequate initial interaction between the 2′P-AMP portion of NADP⁺/H and FNR that provides subsequent conformational changes leading to CTC formation is further confirmed. Stronger interactions between the isoalloxazine and nicotinamide rings might relate with faster HT processes, but exceptions are found upon distortion of the active centre. Thus, within the analyzed FNR variants, there is no strict correlation between the stability of the transient CTCs formation and the rate of the subsequent HT. Kinetic isotope effects suggest that, while in the WT, vibrational enhanced modulation of the active site contributes to the tunnel probability of HT; complexes of some of the active site mutants with the coenzyme hardly allow the relative movement of isoalloxazine and nicotinamide rings along the HT reaction. The architecture of the WT FNR active site precisely contributes to reduce the stacking probability between the isoalloxazine and nicotinamide rings in the catalytically competent complex, modulating the angle and distance between the N5 of the FAD isoalloxazine and the C4 of the coenzyme nicotinamide to values that ensure efficient HT processes.     

Nonresonance Raman study of the flavin cofactor and its interactions in the methylotrophic bacterium W3A1 electron-transfer flavoprotein

Nonresonance Raman spectroscopy has been used to investigate the protein-flavin interactions of the oxidized and anionic semiquinone states of the electron-transfer flavoprotein from the methylotrophic bacteria W3A1 (wETF) in solution. Several unique features of oxidized wETF were revealed from the Raman data. The unusually high frequency of the Raman band for the C(4)=O of the flavin suggests that hydrogen-bonding interactions with the C(4)O are very weak or nonexistent in wETF. In contrast, hydrogen bonding with the C(2)=O is one of the strongest among the flavoproteins investigated thus far. According to the crystal structure, the side-chain hydroxyl group of alphaSer254 serves as a hydrogen bond donor to the N(5) atom in the oxidized flavin cofactor in wETF. The replacement of alphaSer254 by cysteine by site-directed mutagenesis resulted in shifts in N(5)-relevant Raman bands in both the oxidized and anionic semiquinone states of the protein. These results confirm the presence of the hydrogen-bonding interaction at N(5) that is evident in the crystal structure of the oxidized protein and that it persists in the one-electron reduced state. The data suggest that these bands can serve as useful Raman markers for the N(5) interactions in both oxidation states of flavoproteins. The wETF displays unusually low frequencies of flavin ring I (o-xylene ring) relevant bands, which suggests a ring I microenvironment different from most of the other flavoproteins. As indicated by Raman data, the alphaS254C mutation changed the environment of ring I, perhaps as the consequence of changes in the mobility of the FAD domain of wETF. These unusual flavin-protein interactions may be associated with the unique redox properties of wETF.     

Crystal structure of reduced thioredoxin reductase from Escherichia coli: structural flexibility in the isoalloxazine ring of the flavin adenine dinucleotide cofactor

Catalysis by thioredoxin reductase (TrxR) from Escherichia coli requires alternation between two domain arrangements. One of these conformations has been observed by X-ray crystallography (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800-816). This form of TrxR, denoted FO, permits the reaction of enzyme-bound reduced FAD with a redox-active disulfide on TrxR. As part of an investigation of conformational changes and intermediates in catalysis by TrxR, an X-ray structure of the FO form of TrxR with both the FAD and active site disulfide reduced has been determined. Reduction after crystallization resulted in significant local conformation changes. The isoalloxazine ring of the FAD cofactor, which is essentially planar in the oxidized enzyme, assumes a 34 degree "butterfly" bend about the N(5)-N(10) axis in reduced TrxR. Theoretical calculations reported by others predict ring bending of 15-28 degrees for reduced isoalloxazines protonated at N(1). The large bending in reduced TrxR is attributed in part to steric interactions between the isoalloxazine ring and the sulfur of Cys138, formed by reduction of the active site disulfide, and is accompanied by changes in the positions and interactions of several of the ribityl side-chain atoms of FAD. The bending angle in reduced TrxR is larger than that for any flavoprotein in the Protein Data Bank. Distributions of bending angles in published oxidized and reduced flavoenzyme structures are different from those found in studies of free flavins, indicating that the protein environment has a significant effect on bending.     

Structure and Catalytic Mechanism of 3-Ketosteroid-{Delta}4-(5α)-dehydrogenase from Rhodococcus jostii RHA1 Genome

3-Ketosteroid Δ4-(5α)-dehydrogenases (Δ4-(5α)-KSTDs) are enzymes that introduce a double bond between the C4 and C5 atoms of 3-keto-(5α)-steroids. Here we show that the ro05698 gene from Rhodococcus jostii RHA1 codes for a flavoprotein with Δ4-(5α)-KSTD activity. The 1.6 Å resolution crystal structure of the enzyme revealed three conserved residues (Tyr-319, Tyr-466, and Ser-468) in a pocket near the isoalloxazine ring system of the FAD co-factor. Site-directed mutagenesis of these residues confirmed that they are absolutely essential for catalytic activity. A crystal structure with bound product 4-androstene-3,17-dione showed that Ser-468 is in a position in which it can serve as the base abstracting the 4β-proton from the C4 atom of the substrate. Ser-468 is assisted by Tyr-319, which possibly is involved in shuttling the proton to the solvent. Tyr-466 is at hydrogen bonding distance to the C3 oxygen atom of the substrate and can stabilize the keto-enol intermediate occurring during the reaction. Finally, the FAD N5 atom is in a position to be able to abstract the 5α-hydrogen of the substrate as a hydride ion. These features fully explain the reaction catalyzed by Δ4-(5α)-KSTDs.     

The F420-Reducing [NiFe]-Hydrogenase Complex from Methanothermobacter marburgensis, the First X-ray Structure of a Group 3 Family Member

The reversible redox reaction between coenzyme F₄₂₀ and H₂ to F₄₂₀H₂ is catalyzed by an F₄₂₀-reducing [NiFe]-hydrogenase (FrhABG), which is an enzyme of the energy metabolism of methanogenic archaea. FrhABG is a group 3 [NiFe]-hydrogenase with a dodecameric quaternary structure of 1.25 MDa as recently revealed by high-resolution cryo-electron microscopy. We report on the crystal structure of FrhABG from Methanothermobacter marburgensis at 1.7 Å resolution and compare it with the structures of group 1 [NiFe]-hydrogenases, the only group structurally characterized yet. FrhA is similar to the large subunit of group 1 [NiFe]-hydrogenases regarding its core structure and the embedded [NiFe]-center but is different because of the truncation of ca 160 residues that results in similar but modified H₂ and proton transport pathways and in suitable interfaces for oligomerization. The small subunit FrhG is composed of an N-terminal domain related to group 1 enzymes and a new C-terminal ferredoxin-like domain carrying the distal and medial [4Fe-4S] clusters. FrhB adopts a novel fold, binds one [4Fe-4S] cluster as well as one FAD in a U-shaped conformation and provides the F₄₂₀-binding site at the Si-face of the isoalloxazine ring. Similar electrochemical potentials of both catalytic reactions and the electron-transferring [4Fe-4S] clusters, determined to be E°′ ≈ − 400 mV, are in full agreement with the equalized forward and backward rates of the FrhABG reaction. The protein might contribute to balanced redox potentials by the aspartate coordination of the proximal [4Fe-4S] cluster, the new ferredoxin module and a rather negatively charged FAD surrounding.     

Structures of NADH and CH3-H4folate complexes of Escherichia coli methylenetetrahydrofolate reductase reveal a spartan strategy for a ping-pong reaction

Methylenetetrahydrofolate reductases (MTHFRs; EC 1.7.99.5) catalyze the NAD(P)H-dependent reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using flavin adenine dinucleotide (FAD) as a cofactor. The initial X-ray structure of Escherichia coli MTHFR revealed that this 33-kDa polypeptide is a (betaalpha)(8) barrel that aggregates to form an unusual tetramer with only 2-fold symmetry. Structures of reduced enzyme complexed with NADH and of oxidized Glu28Gln enzyme complexed with CH(3)-H(4)folate have now been determined at resolutions of 1.95 and 1.85 A, respectively. The NADH complex reveals a rare mode of dinucleotide binding; NADH adopts a hairpin conformation and is sandwiched between a conserved phenylalanine, Phe223, and the isoalloxazine ring of FAD. The nicotinamide of the bound pyridine nucleotide is stacked against the si face of the flavin ring with C4 adjoining the N5 of FAD, implying that this structure models a complex that is competent for hydride transfer. In the complex with CH(3)-H(4)folate, the pterin ring is also stacked against FAD in an orientation that is favorable for hydride transfer. Thus, the binding sites for the two substrates overlap, as expected for many enzymes that catalyze ping-pong reactions, and several invariant residues interact with both folate and pyridine nucleotide substrates. Comparisons of liganded and substrate-free structures reveal multiple conformations for the loops beta2-alpha2 (L2), beta3-alpha3 (L3), and beta4-alpha4 (L4) and suggest that motions of these loops facilitate the ping-pong reaction. In particular, the L4 loop adopts a "closed" conformation that allows Asp120 to hydrogen bond to the pterin ring in the folate complex but must move to an "open" conformation to allow NADH to bind.     

Electrostatic control of the isoalloxazine environment in the two-electron reduced states of yeast glutathione reductase

The resonance Raman spectra of the oxidized and two-electron reduced forms of yeast glutathione reductase are reported. The spectra of the oxidized enzyme indicate a low electron density for the isoalloxazine ring. As far as the two-electron reduced species are concerned, the spectral comparison of the NADPH-reduced enzyme with the glutathione- or dithiothreitol-reduced enzyme shows significant frequency differences for the flavin bands II, III, and VII. The shift of band VII was correlated with a change in steric or electronic interaction of the hydroxyl group of a conserved Tyr with the N(10)-C(10a) portion of the isoalloxazine ring. Upward shifts of bands II and III observed for the glutathione- or dithiothreitol-reduced enzyme indicate both a slight change in isoalloxazine conformation and a hydrogen bond strengthening at the N(1) and/or N(5) site(s). The formation of a mixed disulfide intermediate tends to slightly decrease the frequency of bands II, III, X, XI, and XIV. To account for the different spectral features observed for the NADPH- and glutathione-reduced species, several possibilities have been examined. In particular, we propose a hydrogen bonding modulation at the N(5) site of FAD through a variable conformation of an ammonium group of a conserved Lys residue. Changes in N(5)(flavin)-protein interaction in the two-electron reduced forms of glutathione reductase are discussed in relation to a plausible mechanism of the regulation of the enzyme activity via a variable redox potential of FAD.     

Location of lysyl residues at the allosteric site of fructose 1,6-bisphosphatase

Various flavin analogs were used as alternate substrates or competitive inhibitors to characterize the FMN binding sites of the NADH- and NADPH-specific FMN oxidoreductases from Beneckea harveyi. Several polyhydroxyl compounds were found to be poor competitive inhibitors for the FMN sites of these enzymes. The FMN binding sites of the two enzymes were found to be quite similar. The NADH:FMN oxidoreductase binds FMN exclusively through the isoalloxazine ring. The methyl groups at positions 7 and 8 contribute significantly to this binding. Utilizing lumichrome as a competitive inhibitor of the FMN binding site and AMP as a competitive inhibitor of the NADH binding site, we were able to determine that the NADH:FMN oxidoreductase forms an active ternary complex with NADH binding first in an ordered mechanism. The NADPH oxidoreductase also binds FMN primarily through the isoalloxazine ring. Unlike their participation in reaction with the NADH-specfic enzyme, the methyl groups at positions 7 and 8 are not involved in binding. There was no significant binding of the ribityl phosphate moiety with either enzyme. Both enzymes have lower K_m values for lumiflavin than FMN.     

Structure and mutation analysis of archaeal geranylgeranyl reductase

The crystal structure of geranylgeranyl reductase (GGR) from Sulfolobus acidocaldarius was determined in order to elucidate the molecular mechanism of the catalytic reaction. The enzyme is a flavoprotein and is involved in saturation of the double bonds on the isoprenoid moiety of archaeal membranes. The structure determined in this study belongs to the p-hydroxybenzoate hydroxylase family in the glutathione reductase superfamily. GGR functions as a monomer and is divided into the FAD-binding, catalytic and C-terminal domains. The catalytic domain has a large cavity surrounded by a characteristic YxWxFPx_7-8GxG motif and by the isoalloxazine ring of an FAD molecule. The cavity holds a lipid molecule, which is probably derived from Escherichia coli cells used for over-expression. One of the two forms of the structure clarifies the presence of an anion pocket holding a pyrophosphate molecule, which might anchor the phosphate head of the natural ligands. Mutational analysis supports the suggestion that the three aromatic residues of the YxWxFPx_7-8GxG motif hold the ligand in the appropriate position for reduction. Cys47, which is widely conserved in GGRs, is located at the si-side of the isoalloxazine ring of FAD and is shown by mutational analysis to be involved in catalysis. The catalytic cycle, including the FAD reducing factor binding site, is proposed on the basis of the detailed analysis of the structure.     

Analysis of the structure of the flavin-binding sites of flavocytochrome P450 BM3 using surface enhanced resonance Raman scattering

Flavocytochrome P450 BM3, an FMN-deficient mutant (G570 D), the component reductase and an FAD-containing domain were studied using surface enhanced resonance Raman scattering (SERRS). They were compared to spectra obtained from the free flavins FAD and FMN. For the holoenzyme and reductase domain, FMN is displaced during SERRS analysis. However, studies with the G570 D mutant indicate that FAD is retained in its active site. Analysis of SERRS frequencies and intensities provides information on the nature of the flavin binding site and the planarity of the ring, and enables an interpretation of the hydrogen bonding environment around ring III of the isoalloxazine moiety. Hydrogen bonding is strong at N₃–H, C₂=O and C₄=O, but weak at N₅. Structural alteration of the FAD domain of P450 BM3 is caused by removal of the FMN-binding domain. Further, the hydrogen bond at N₃–H is lost and that at C₂=O is weakened and the isoalloxazine ring system in the FAD domain appears to adopt a more planar arrangement. Alterations in the environment of the FAD in its isolated domain are likely to relate to changes in the redox properties and suggest a close structural interplay of FAD with the FMN-binding domain in intact flavocytochrome P450 BM3. Received: 5 August 1998 / Revised version: 11 February 1999 / Accepted: 15 February 1999     

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (Etf_Af) and butyryl-CoA dehydrogenase (Bcd_Af) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. Etf_Af contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The Etf_Af-NAD⁺ complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH⁻ is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH⁻ by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD^⨪, which donates this electron further to Dh-FAD of Bcd_Af after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH^•, immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH⁻ that converts crotonyl-CoA to butyryl-CoA.