Refined crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii at 2.2 A resolution. A comparison with the structure of glutathione reductase

The structure of lipoamide dehydrogenase from Azotobacter vinelandii has been refined by the molecular dynamics technique to an R-factor of 19.8% at 2.2 A resolution. In the final model, the root-mean-square deviation from ideality is 0.02 A for bond lengths and 3.2 degrees for bond angles. The asymmetric unit comprises two subunits, each consisting of 466 amino acid residues and the prosthetic group FAD, plus 512 solvent molecules. The last ten amino acid residues of both chains are not visible in the electron density distribution and they are probably disordered. The operation required to superimpose the two chains forming the dimer is a rotation of exactly 180 degrees with no translation component. The final model shows the two independently refined subunits to be very similar, except for six loops located at the surface of the molecule. The structure of each subunit of the enzyme consists of four domains with the catalytic centre located at the subunit interface. The reactive disulphide bridge, 48-53, is oxidized with S gamma of Cys53 located 3.5 A away from carbon C-4a of the isoalloxazine ring. The side-chain of His450' points its N epsilon 2 towards S gamma of Cys48 and is hydrogen bonded to the carboxylate of Glu455'. The FAD is bound in an extended conformation and the isoalloxazine ring is not completely planar with an angle between the pteridine and the benzene ring of 7.3 degrees in the first subunit and of 12.1 degrees in the second one. The overall folding of lipoamide dehydrogenase is very similar to that of glutathione reductase. However, a comparison of the two enzymes, which have only 26% sequence identity, reveals significant conformational differences. These concern the tertiary as well as the quaternary structure of the two molecules. In each subunit of lipoamide dehydrogenase the NAD-binding domain and the interface domain appear to be differently oriented with respect to the FAD-binding domain by 7.1 degrees and 7.8 degrees, respectively. The interface domain contains, in addition, major changes in tertiary structure. Furthermore, the two subunits forming the dimer appear to be shifted with respect to each other by more than 4 A, when the lipoamide dehydrogenase dimer is compared with that of glutathione reductase. In spite of all these changes at the tertiary and quaternary level the active sites of the enzymes, which occur at the dimer interface, appear to be remarkably similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Picosecond fluorescence lifetime of the coenzyme of D-amino acid oxidase

Conformational difference surrounding the coenzyme, FAD, of D-amino acid oxidase (D-amino-acid:O2 oxidoreductase (deaminating), EC 1.4.3.3) between its monomeric and dimeric forms were examined by observing fluorescence of FAD. The fluorescence lifetimes of the coenzyme was measured directly with a mode-locked Nd:YAG laser and a streak camera in picosecond region. The values of lifetime of FAD fluorescence in the monomer and dimer were 130 +/- 20 ps and 40 +/- 10 ps, respectively. The relative quantum yield of the fluorescence of FAD combined with the protein to that of free FAD depended on the concentration of the enzyme; it was higher at lower concentration. Comparing the lifetime with relative quantum yield of FAD combined with the protein, it is concluded that the fluorescence is quenched mostly by a dynamic process. These results indicate that the distance between the isoalloxazine nucleus and a quencher is nearer in the dimer than in the monomer.     

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.     

Crystal structure of the flavin reductase component (HpaC) of 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8: Structural basis for the flavin affinity

The two-component enzyme, 4-hydroxyphenylacetate 3-monooxygenase, catalyzes the conversion of 4-hydroxyphenylacetate to 3,4-dihydroxyphenylacetate. In the overall reaction, the oxygenase component (HpaB) introduces a hydroxyl group into the benzene ring of 4-hydroxyphenylacetate using molecular oxygen and reduced flavin, while the reductase component (HpaC) provides free reduced flavins for HpaB. The crystal structures of HpaC from Thermus thermophilus HB8 in the ligand-free form, the FAD-containing form, and the ternary complex with FAD and NAD(+) were determined. In the ligand-free form, two large grooves are present at the dimer interface, and are occupied by water molecules. A structural analysis of HpaC containing FAD revealed that FAD has a low occupancy, indicating that it is not tightly bound to HpaC. This was further confirmed in flavin dissociation experiments, showing that FAD can be released from HpaC. The structure of the ternary complex revealed that FAD and NAD(+) are bound in the groove in the extended and folded conformation, respectively. The nicotinamide ring of NAD(+) is sandwiched between the adenine ring of NAD(+) and the isoalloxazine ring of FAD. The distance between N5 of the isoalloxazine ring and C4 of the nicotinamide ring is about 3.3 A, sufficient to permit hydride transfer. The structures of these three states are essentially identical, however, the side chains of several residues show small conformational changes, indicating an induced fit upon binding of NADH. Inactivity with respect to NADPH can be explained as instability of the binding of NADPH with the negatively charged 2'-phosphate group buried inside the complex, as well as a possible repulsive effect by the dipole of helix alpha1. A comparison of the binding mode of FAD with that in PheA2 from Bacillus thermoglucosidasius A7, which contains FAD as a prosthetic group, reveals remarkable conformational differences in a less conserved loop region (Gly83-Gly94) involved in the binding of the AMP moiety of FAD. These data suggest that variations in the affinities for FAD in the reductases of the two-component flavin-diffusible monooxygenase family may be attributed to difference in the interaction between the AMP moiety of FAD and the less conserved loop region which possibly shows structural divergence.     

FAD analogues as prosthetic groups of human glutathione reductase. Properties of the modified enzyme species and comparisons with the active site structure

Human glutathione reductase (NADPH + GSSG + H+ in equilibrium with NADP+ + 2 GSH) is a suitable enzyme for correlating spectroscopic properties and chemical reactivities of protein-bound FAD analogues with structural data. FAD, the prosthetic group of the enzyme, was replaced by FAD analogues, which were modified at the positions 8, 1, 2, 4, 5 and 6, respectively, of the isoalloxazine ring. When compared with a value of 100% for native glutathione reductase, the specific activities of most enzyme species ranged from 40% to 17%, in the order of the prosthetic groups 8-mercapto-FAD greater than 8-azido-FAD = 8-F-FAD = 8-C1-FAD greater than 4-thio-FAD = 1-deaza-FAD greater than 2-thio-FAD. The enzymic activities indicate a correct orientation of the bound analogues. The enzyme species containing 5-deaza-FAD and 6-OH-FAD, respectively, had no more glutathione reductase activity than the FAD-free apoenzyme. 5-Deaza-FAD X glutathione reductase was crystallized for X-ray diffraction analysis. Detailed studies were focussed on position 8 of the flavin. 8-Cl-FAD X glutathione reductase and 8-F-FAD X glutathione reductase reacted only poorly with HS- to give 8-mercapto-FAD X glutathione reductase, which suggests that the region around Val61 hinders the halogen anion from leaving the tetrahedral intermediate. Other experiments showed that position 8 is accessible to certain solvent-borne reagents. 8-Mercapto-FAD X glutathione reductase, for instance, reacted readily and stoichiometrically with the thiol reagent methylmethanethiosulfonate. 8-Mercapto-FAD X glutathione reductase does not exhibit a long wavelength charge transfer absorption band upon reduction, as it is the case for the 2-electron-reduced FAD-containing enzyme. This behaviour indicates that the charge transfer interaction between flavin and the thiolate of Cys63 in the native enzyme is not per se essential for catalysis. The absorption spectrum of the blue anionic 8-mercapto-FAD bound to glutathione reductase suggests that the protein concurs to the stabilization of a negative charge in the pyrimidine subnucleus. In light of the protein structure this effect is attributed to the dipole moment of alpha-helix 338-354 which starts out close to the N(1)/C(2)/O(2 alpha) region of the flavin. 1-Deaza-FAD binds as tightly as FAD to the apoenzyme. The resulting holoenzyme was found to be enzymically active but structurally unstable. In this respect 1-deaza-FAD . glutathione reductase mimics the properties of the enzyme species found in inborn glutathione reductase deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformational change of Arabidopsis thaliana thioredoxin reductase after binding of pyridine nucleotide and thioredoxin

We have found that the binding of NADP+ (Kd = 0.86+/-0.11 microM) enhanced the FAD fluorescence of Arabidopsis thaliana NADPH:thioredoxin reductase (TR, EC 1.6.4.5) by 2 times, whereas the binding of 3-aminopyridine adenine dinucleotide phosphate (AADP+) (Kd < 0.1 microM) quenched the fluorescence by 20%. Thioredoxin (TRX) also enhanced the FAD fluorescence by 35%. The Kd of TR-NADP+ and TR-AADP+ complexes did not change in the presence of 45 microM TRX. Our findings imply that the binding of NADP+ and AADP+ at the NADP(H)-binding site of A. thaliana TR, and/or the binding of TRX in the vicinity of the catalytic disulfide increase the content of fluorescent FR conformer (NADP(H)-binding site adjacent to flavin). The different effects of NADP+ and AADP+ on FAD fluorescence intensity may be explained by the superposition of two opposite factors: i) increased content of fluorescent FR conformer upon binding of NADP+ or AADP+; ii) quenching of FAD fluorescence by electron-donating 3-aminopyridinium ring of AADP+.     

Properties of rabbit liver glutathione reductase reconstituted with FAD analogs

The FAD binding site of rabbit liver glutathione reductase has been explored by reconstitution of the apoprotein with several FAD analogs modified in the isoalloxazine ring. The apoglutathione reductase binds the p-quinoid form of 8-mercapto-FAD, suggesting that the protein stabilizes a negative charge in the -N1-C2 = O position of the pyrimidine subnucleus. The main absorption peak in the visible spectrum of the 8-mercapto-FAD-enzyme is at 585 nm; treatment of the reconstituted protein with reducing agents of disulfide groups induces a reversible hypochromic shift of 20 nm of the peak. Thus, in 8-mercapto-FAD-glutathione reductase, the oxidation-reduction state of the active center disulfide can be monitored. The chemical reactivity toward methylmethanethiosulfonate and iodoacetamide of the 8-mercapto-FAD-enzyme shows that the flavin position 8 is freely accessible to solvent. However, position 2 is buried within the protein molecule as judged from the lack of reactivity of the 2-thio-FAD-enzyme with methylmethanethiosulfonate. Hydrogen peroxide reacts slowly with both 2-thio-FAD-enzyme and native glutathione reductase, yielding inactive enzyme with a modified spectrum; the prosthetic group is still protein bound. Differences in the active site of the rabbit liver enzyme compared to the human erythrocyte glutathione reductase are evidenced by use of FAD analogs: the peaks of reconstituted liver enzymes are shifted about 10 nm toward longer wavelengths.     

NADPH-cytochrome P450 oxidoreductase. Structural basis for hydride and electron transfer

NADPH-cytochrome P450 oxidoreductase catalyzes transfer of electrons from NADPH, via two flavin cofactors, to various cytochrome P450s. The crystal structure of the rat reductase complexed with NADP(+) has revealed that nicotinamide access to FAD is blocked by an aromatic residue (Trp-677), which stacks against the re-face of the isoalloxazine ring of the flavin. To investigate the nature of interactions between the nicotinamide, FAD, and Trp-677 during the catalytic cycle, three mutant proteins were studied by crystallography. The first mutant, W677X, has the last two C-terminal residues, Trp-677 and Ser-678, removed; the second mutant, W677G, retains the C-terminal serine residue. The third mutant has the following three catalytic residues substituted: S457A, C630A, and D675N. In the W677X and W677G structures, the nicotinamide moiety of NADP(+) lies against the FAD isoalloxazine ring with a tilt of approximately 30 degrees between the planes of the two rings. These results, together with the S457A/C630A/D675N structure, allow us to propose a mechanism for hydride transfer regulated by changes in hydrogen bonding and pi-pi interactions between the isoalloxazine ring and either the nicotinamide ring or Trp-677 indole ring. Superimposition of the mutant and wild-type structures shows significant mobility between the two flavin domains of the enzyme. This, together with the high degree of disorder observed in the FMN domain of all three mutant structures, suggests that conformational changes occur during catalysis.     

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.     

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.     

Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 A resolution. Analysis of the enzyme-substrate and enzyme-product complexes

Using synchrotron radiation, the X-ray diffraction intensities of crystals of p-hydroxy-benzoate hydroxylase, complexed with the substrate p-hydroxybenzoate, were measured to a resolution of 1.9 A. Restrained least-squares refinement alternated with rebuilding in electron density maps yielded an atom model of the enzyme-substrate complex with a crystallographic R-factor of 15.6% for 31,148 reflections between 6.0 and 1.9 A. A total of 330 solvent molecules was located. In the final model, only three residues have deviating phi-psi angle combinations. One of them, the active site residue Arg44, has a well-defined electron density and may be strained to adopt this conformation for efficient catalysis. The mode of binding of FAD is distinctly different for the different components of the coenzyme. The adenine ring is engaged in three water-mediated hydrogen bonds with the protein, while making only one direct hydrogen bond with the enzyme. The pyrophosphate moiety makes five water-mediated versus three direct hydrogen bonds. The ribityl and ribose moieties make only direct hydrogen bonds, in all cases, except one, with side-chain atoms. The isoalloxazine ring also makes only direct hydrogen bonds, but virtually only with main-chain atoms. The conformation of FAD in p-hydroxybenzoate hydroxylase is strikingly similar to that in glutathione reductase, while the riboflavin-binding parts of these two enzymes have no structural similarity at all. The refined 1.9 A structure of the p-hydroxybenzoate hydroxylase-substrate complex was the basis of further refinement of the 2.3 A structure of the enzyme-product complex. The result was a final R-factor of 16.7% for 14,339 reflections between 6.0 and 2.3 A and an improved geometry. Comparison between the complexes indicated only small differences in the active site region, where the product molecule is rotated by 14 degrees compared with the substrate in the enzyme-substrate complex. During the refinements of the enzyme-substrate and enzyme-product complexes, the flavin ring was allowed to bend or twist by imposing planarity restraints on the benzene and pyrimidine ring, but not on the flavin ring as a whole. The observed angle between the benzene ring and the pyrimidine ring was 10 degrees for the enzyme-substrate complex and 19 degrees for the enzyme-product complex. Because of the high temperature factors of the flavin ring in the enzyme-product complex, the latter value should be treated with caution. Six out of eight peptide residues near the flavin ring are oriented with their nitrogen atom pointing towards the ring.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electronic transitions in the isoalloxazine ring and orientation of flavins in model membranes studied by polarized light spectroscopy.

The orientation of flavin mononucleotide (FMN) in model membranes and the directions of the transition moments of the first three bands in the electronic absorption spectrum of the oxidized form of the isoalloxazine ring have been determined by means of linear dichroism and polarized fluorescence spectroscopy. Measured counterclockwise relative to the axis connecting the two nitrogens in the central ring (considered positive when going in the direction from -CN less than to greater than or equal to N), these angles are 58 +/- 4 degrees (450-nm band), 97 +/- 3 degrees (350-nm band), and 119 +/- 2 degrees (260-nm band).     

Properties of lipoamide dehydrogenase altered by site-directed mutagenesis at a key residue (I184Y) in the pyridine nucleotide binding domain.

The binding of pyridine nucleotide to human erythrocyte glutathione reductase, an enzyme of known three-dimensional structure, requires some movement of the side chain of Tyr197. Moreover, this side chain lies very close to the isoalloxazine ring of the FAD cofactor. The analogous residue, Ile184, in the homologous enzyme Escherichia coli lipoamide dehydrogenase has been altered by site-directed mutagenesis to a tyrosine residue (I184Y) [Russell, G. C., Allison, N., Williams, C. H., Jr., & Guest, J.R. (1989) Ann. N.Y. Acad. Sci. 573, 429-431]. Characterization of the altered enzyme shows that the rate of the pyridine nucleotide half-reaction has been markedly reduced and that the spectral properties have been changed to mimic those of glutathione reductase. Therefore, Ile184 is shown to be an important residue in modulating the properties of the flavin in lipoamide dehydrogenase. Turnover in the dihydrolipoamide/NAD+ reaction is decreased by 10-fold and in the NADH/lipoamide reaction by 2-fold in I184Y lipoamide dehydrogenase. The oxidized form of I184Y shows remarkable changes in the fine structure of the visible absorption and circular dichroism spectra and also shows nearly complete quenching of FAD fluorescence. The spectral properties of the altered enzyme are thus similar to those of glutathione reductase and very different from those of wild-type lipoamide dehydrogenase. On the other hand, spectral evidence does not reveal any change in the amount of charge-transfer stabilization at the EH2 level. Stopped-flow data indicate that, in the reduction of I184Y by NADH, the first step, reduction of the flavin, is only slightly slowed but the subsequent two-electron transfer to the disulfide is markedly inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rabbit liver glutathione reductase. Purification and properties

Hepatic glutathione reductase can be obtained in relatively good amounts from rabbit by a procedure which is fairly simple and sufficiently rapid. The purified flavoprotein shows absorbance ratios at 274, 379, and 463 nm of 8.2:0.92:1.0, respectively; the FAD fluorescence is nearly completely quenched by the protein. Gradient ultracentrifugation and sodium dodecyl sulfate gel electrophoresis indicate that the enzyme is a dimer, consisting of subunits of about 56,000 molecular weight; flavin content suggests one FAD per chain. Gel filtration under a variety of conditions, on the other hand, yields a molecular weight in the range 56,000–67,000. It is proposed that rabbit liver glutathione reductase can be active also as monomer. Kinetic parameters of the enzyme have been determined under optimal conditions. The rabbit liver glutathione reductase is, at physiological pH, absolutely specific for NADPH.     

Sequence-structure analysis of FAD-containing proteins

We have analyzed structure-sequence relationships in 32 families of flavin adenine dinucleotide (FAD)-binding proteins, to prepare for genomic-scale analyses of this family. Four different FAD-family folds were identified, each containing at least two or more protein families. Three of these families, exemplified by glutathione reductase (GR), ferredoxin reductase (FR), and p-cresol methylhydroxylase (PCMH) were previously defined, and a family represented by pyruvate oxidase (PO) is newly defined. For each of the families, several conserved sequence motifs have been characterized. Several newly recognized sequence motifs are reported here for the PO, GR, and PCMH families. Each FAD fold can be uniquely identified by the presence of distinctive conserved sequence motifs. We also analyzed cofactor properties, some of which are conserved within a family fold while others display variability. Among the conserved properties is cofactor directionality: in some FAD-structural families, the adenine ring of the FAD points toward the FAD-binding domain, whereas in others the isoalloxazine ring points toward this domain. In contrast, the FAD conformation and orientation are conserved in some families while in others it displays some variability. Nevertheless, there are clear correlations among the FAD-family fold, the shape of the pocket, and the FAD conformation. Our general findings are as follows: (a) no single protein 'pharmacophore' exists for binding FAD; (b) in every FAD-binding family, the pyrophosphate moiety binds to the most strongly conserved sequence motif, suggesting that pyrophosphate binding is a significant component of molecular recognition; and (c) sequence motifs can identify proteins that bind phosphate-containing ligands.     

pH-induced changes in activity and conformation of NADH oxidase from Thermus thermophilus

Thermus thermophilus NADH oxidase (NOX) activity exhibits a bell-shaped pH-dependency with the maximal rate at pH 5.2 and marked inhibition at lower pH. The first pH transition, from pH 7.2 to pH 5.2, results in more than a 2-fold activity increase with protonation of a group with pKa=6.1+/-0.1. The difference in fluorescence of the free and enzyme-bound flavin strongly indicates that the increase in enzyme activity in a pH-dependent manner is related to a protein-cofactor interaction. Only one amino acid residue, His75, has an intrinsic pKa approximately 6.0 and is localized in proximity (<10 A) to N5-N10 of the isoalloxazine ring and, therefore, is able to participate in such an interaction. Solvent acidification leads to the second pH transition from pH 5.2 to 2.0 that results in complete inhibition of the enzyme with protonation of a group with an apparent pKa=4.0+/-0.1. Inactivation of NOX activity at low pH is not caused by large conformational changes in the quaternary structure as judged by intrinsic viscosity and sedimentation velocity experiments. NOX exists as a dimer even as an apoprotein at acidic conditions. There is a strong coupling between the fluorescence of the enzyme-bound flavin and the intrinsic tryptophans, as demonstrated by energy transfer between Trp47 and the isoalloxazine ring of flavin adenine dinucleotide (FAD). The pH-induced changes in intrinsic tryptophan and FAD fluorescence indicate that inhibition of the FAD-binding enzyme at low pH is related to dissociation of the flavin cofactor, due to protonation of its adenine moiety.     

Sequence of thioredoxin reductase from Escherichia coli. Relationship to other flavoprotein disulfide oxidoreductases

The DNA sequence of the Escherichia coli gene encoding thioredoxin reductase has been determined. The predicted protein sequence agrees with an earlier determination of the 17 amino-terminal amino acids and with a fragment of the protein containing the redox-active half-cystines. Similarity between E. coli thioredoxin reductase and other flavoprotein disulfide oxidoreductases is quite limited, but three short segments, two of which are probably involved in FAD and NADPH binding, are highly conserved between thioredoxin reductase, glutathione reductase, dihydrolipoamide dehydrogenase, and mercuric reductase.     

Electron transfer by diflavin reductases

Diflavin reductases are enzymes which emerged as a gene fusion of ferredoxin (flavodoxin) reductase and flavodoxin. The enzymes of this family tightly bind two flavin cofactors, FAD and FMN, and catalyze transfer of the reducing equivalents from the two-electron donor NADPH to a variety of one-electron acceptors. Cytochrome P450 reductase (P450R), a flavoprotein subunit of sulfite reductase (SiR), and flavoprotein domains of naturally occurring flavocytochrome fusion enzymes like nitric oxide synthases (NOS) and the fatty acid hydroxylase from Bacillus megaterium are some of the enzymes of this family. In this review the results of the last decade of research are summarized, and some earlier results are reevaluated as well. The kinetic mechanism of cytochrome c reduction is analyzed in light of other results on flavoprotein interactions with nucleotides and cytochromes. The roles of the binding sites of the isoalloxazine rings of the flavin cofactors and conformational changes of the protein in electron transfer are discussed. It is proposed that minor conformational changes during catalysis can potentiate properties of the redox centers during the catalytic turnover. A function of the aromatic residue that shields the isoalloxazine ring of the FAD is also proposed.     

The structure of NADH peroxidase from Streptococcus faecalis at 3.3 A resolution

NADH peroxidase (EC 1.11.1.1) previously isolated from Streptococcus faecalis 10C1 has been crystallized. The crystal structure has been solved by multiple isomorphous replacement and solvent-flattening at 3.3 A (1 A = 0.1 nm) resolution. The enzyme forms a tetramer consisting of 4 crystallographically related subunits. The monomer chain fold is in general similar to those of glutathione reductase and lipoamide dehydrogenase. FAD binds in the same region and in a similar conformation as in glutathione reductase. The unusual cysteine-sulfenic acid participating in catalysis is located at the isoalloxazine of FAD.     

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