The non-flavin redox center of the streptococcal NADH peroxidase. I. Thiol reactivity and redox behavior in the presence of urea

Unlike the 2-electron-reduced (EH2) forms of the flavoprotein disulfide reductases and mercuric reductase, the native EH2 form of the streptococcal NADH peroxidase is quite refractile toward chemical modification with thiol-specific reagents. In the presence of 1.3 M urea, however, the single thiol of the reduced enzyme reacts with phenylmercuric acetate with a t1/2 of 3 min. This modification abolishes the charge-transfer absorbance band at 540 nm and inactivates the enzyme; the latter effect is shown to be reversed with dithiothreitol. Alkylation of the streptococcal peroxidase with iodo[1-14C]acetamide under reducing conditions in the presence of 8 M guanidine hydrochloride allows the isolation of a single labeled tryptic peptide with the sequence: Gly-Asp-Phe-Ile-Ser-Phe-Leu-Ser-C*ys-Gly-Met-Gln-Leu-Tyr-Leu- Glu-Gly-Lys. This sequence is identical to that previously reported (Poole, L. B., and Claiborne, A. (1988) Biochem. Biophys. Res. Commun. 153, 261-266) for the cysteinyl peptide isolated from the NADH peroxidase labeled metabolically with [35S]cysteine. Careful examination of the physical properties of the streptococcal peroxidase in the presence of 1.3 M urea shows that, while catalytic activity and native structural features are largely retained, the relative potentials of flavin and non-flavin redox centers are dramatically affected. We propose that low concentrations of urea stabilize an intermediate state in the transition between native and denatured forms, which is responsible for the observed changes in both active-site thiol reactivity and in redox properties.     

The streptococcal flavoprotein NADH oxidase. I. Evidence linking NADH oxidase and NADH peroxidase cysteinyl redox centers

The FAD-containing NADH oxidase from Streptococcus faecalis 10C1, which catalyzes the four-electron reduction of O2----2H2O, has been purified by an improved procedure for analyses of its structural and redox properties. The enzyme is apparently a dimer of two identical subunits, each containing 1 mol of FAD. Dithionite reduction of the enzyme proceeds in two distinct phases corresponding to approximately 0.5 and 1.1 eq/FAD, respectively. Thiol assays of the NADH oxidase, reduced anaerobically with 1 eq of NADH/FAD prior to denaturation, are consistent with the presence of a single redox-active cysteinyl residue/subunit. Analysis of the cysteinyl peptides of the oxidase, identified in tryptic digests of the enzyme labeled metabolically with [35S]cysteine, reveals a sequence which is closely related to the redox-active cysteinyl peptide sequence recently determined for the streptococcal flavoprotein NADH peroxidase. A second cysteinyl peptide sequence, when aligned with residues 3-17 of the peroxidase NH2-terminal sequence, reveals identity in 7 of 15 positions and satisfies several of the criteria described for ADP-binding structures. Additional probes of the structural and redox properties of the NADH oxidase, including visible circular dichroism spectroscopy and sensitivity to inactivation by hydrogen peroxide, provide further evidence for a fundamental structural connection between flavin-dependent NADH oxidase and peroxidase functions.     

Catalytic properties of streptococcal NADH oxidase containing artificial flavins.

The flavoprotein NADH oxidase from Streptococcus faecalis 10C1, which catalyzes the tetravalent reduction of O2-->2H2O, has been purified as the apoenzyme to allow reconstitution studies with both native and artificial flavins. Turnover numbers for the enzyme containing 1-deaza-, 2-thio-, and 4-thio-FAD range from 51 to 4% of that of the native FAD enzyme; these reconstituted oxidases also catalyze the four-electron reduction of oxygen. Dithionite and NADH titrations of the native FAD oxidase require 1.7 eq of reductant/FAD and follow spectral courses very similar to those previously reported for the purified holoenzyme. Azide is a linear mixed-type inhibitor with respect to NADH, and dithionite titrations in the presence of azide yield significant stabilization of the neutral blue semiquinone. Redox stoichiometries for the oxidase containing modified flavins range from 1.1 to 1.4 eq of reductant/FAD. Spectrally distinct reduced enzyme.NAD+ complexes result with all but the 2-thio-FAD enzyme on titration with NADH. The reduced 4-thio-FAD oxidase shows little or no evidence of desulfurization to native FAD on reduction and reoxidation. Both the 8-mercapto- (E'o = -290 mV) and 8-hydroxy-FAD (E'o = -335 mV) oxidase are readily reduced by excess NADH. These results offer a further basis for analysis of the active-site structure and oxygen reactivity of this unique flavoprotein oxidase.     

Interactions of pyridine nucleotides with redox forms of the flavin-containing NADH peroxidase from Streptococcus faecalis

The flavin-containing NADH peroxidase of Streptococcus faecalis 10C1, which catalyzes the reaction: NADH + H+ + H2O2----NAD+ + 2H2O, has been purified to homogeneity in our laboratory for analyses of both its structure and redox behavior. Our findings indicate that the enzyme is a tetramer of four apparently identical subunits (Mr = 46,000/subunit), each containing one FAD coenzyme and a second non-flavin, nonmetal redox center. There is no evidence of nonequivalence among the flavins. Dithionite reduction of the enzyme occurs in two steps, with end points of 0.96 and 2.05 eq/FAD. The first step generates a two-electron reduced form of the enzyme (EH2) which is spectrally identical with that generated by aerobic addition of NADH. Our studies suggest that the long-wavelength absorbance band (lambda max approximately 540 nm) exhibited by this form results from charge-transfer interaction between the reduced non-flavin redox center and the oxidized flavin. A second type of long-wavelength charge-transfer absorbance band (lambda max approximately 770 nm) is generated on anaerobic addition of 1 eq of NADH to EH2 and results from interaction between oxidized FAD and the reduced pyridine nucleotide. Either the EH2 X NAD+ or the EH2 X NAD+ X NADH forms may be involved in the catalytic mechanism of the enzyme, as both are reactive with hydrogen peroxide.     

The streptococcal flavoprotein NADH oxidase. II. Interactions of pyridine nucleotides with reduced and oxidized enzyme forms

Anaerobic addition of 0.5 eq of NADH/FAD to the streptococcal NADH oxidase produces a redox form spectrally similar to that obtained with 0.5 eq of dithionite/FAD. The second phase of the titration, however, in addition to reducing the flavin with 1 eq of NADH/FAD, leads to the appearance of a long-wavelength absorbance band centered at 725 nm. Reductive titrations of the enzyme with 3-acetylpyridine-adenine dinucleotide, which has a redox potential 72 mV more positive than that of NADH, yield a similar reduced enzyme species. Dithionite reduction of the NADH oxidase followed by titration with NAD+ partially mimics the long-wavelength absorbance of the NADH-reduced enzyme but also leads to the oxidation of 1 FADH2/dimer. NADH is not formed, however, and a similar result is obtained when the dithionite-reduced oxidase is titrated with the nonreducible substrate analog 3-aminopyridine-adenine dinucleotide. These data indicate that the FADH2 oxidation observed is intramolecular and suggest that the active centers of the two apparently identical subunits/dimer are not equivalent. These results also demonstrate that bound pyridine nucleotides can modulate the redox manifold of the NADH oxidase and, when taken together with the effects of these ligands on pre-steady-state behavior, suggest an important regulatory aspect of the catalytic redox function of this unique flavoprotein.     

Differences in protein structure of xanthine dehydrogenase and xanthine oxidase revealed by reconstitution with flavin active site probes

The native flavin, FAD, was removed from chicken liver xanthine dehydrogenase and milk xanthine oxidase by incubation with CaCl2. The deflavoenzymes, still retaining their molybdopterin and iron-sulfur prosthetic groups, were reconstituted with a series of FAD derivatives containing chemically reactive or environmentally sensitive substituents in the isoalloxazine ring system. The reconstituted enzymes containing these artificial flavins were all catalytically active. With both the chicken liver dehydrogenase and the milk oxidase, the flavin 8-position was found to be freely accessible to solvent. The flavin 6-position was also freely accessible to solvent in milk xanthine oxidase, but was significantly less exposed to solvent in the chicken liver dehydrogenase. Pronounced differences in protein structure surrounding the bound flavin were indicated by the spectral properties of the two enzymes reconstituted with flavins containing ionizable -OH or -SH substituents at the flavin 6- or 8-positions. Milk xanthine oxidase either displayed no preference for binding of the neutral or anionic flavin (8-OH-FAD) or a slight preference for the anionic form of the flavin (6-hydroxy-FAD, 6-mercapto-FAD, and possibly 8-mercapto-FAD). On the other hand, the chicken liver dehydrogenase had a dramatic preference for binding the neutral (protonated) forms of all four flavins, perturbing the pK of the ionizable substituent greater than or equal to 4 pH units. These results imply the existence of a strong negative charge in the flavin binding site of the dehydrogenase, which is absent in the oxidase.     

Equilibrium analyses of the active-site asymmetry in enterococcal NADH oxidase: role of the cysteine-sulfenic acid redox center

Recent studies [Mallett, T. C., and Claiborne, A. (1998) Biochemistry 37, 8790-8802] of the O2 reactivity of C42S NADH oxidase (O2 --> H2O2) revealed an asymmetric mechanism in which the two FADH2.NAD+ per reduced dimer display kinetic inequivalence. In this report we provide evidence indicating that the fully active, recombinant wild-type oxidase (O2 --> 2H2O) displays thermodynamic inequivalence between the two active sites per dimer. Using NADPH to generate the free reduced wild-type enzyme (EH2'/EH4), we have shown that NAD+ titrations lead to differential behavior as only one FADH2 per dimer binds NAD+ tightly to give the charge-transfer complex. The second FADH2, in contrast, transfers its electrons to the single Cys42-sulfenic acid (Cys42-SOH) redox center, which remains oxidized during the reductive titration. Titrations of the reduced NADH oxidase with oxidized 3-acetylpyridine and 3-aminopyridine adenine dinucleotides further support the conclusion that the two FADH2 per dimer in wild-type enzyme can be described as distinct "charge-transfer" and "electron-transfer" sites, with the latter site giving rise to either intramolecular (Cys42-SOH) or bimolecular (pyridine nucleotide) reduction. The reduced C42S mutant is not capable of intramolecular electron transfer on binding pyridine nucleotides, thus confirming that the Cys42-SOH center is in fact the source of the redox asymmetry observed with wild-type oxidase. These observations on the role of Cys42-SOH in the expression of thermodynamic inequivalence as observed in wild-type NADH oxidase complement the previously described kinetic inequivalence of the C42S mutant; taken together, these results provide the overlapping framework for an alternating sites cooperativity model of oxidase action.     

The reaction of 8-mercaptoflavins and flavoproteins with sulfite. Evidence for the role of an active site arginine in D-amino acid oxidase

This work presents strong evidence that the role of the active site arginine in D-amino acid oxidase is to act as a positively charged group interacting with the flavin N(1)-C(2) = 0 locus. Modification with cyclohexanedione, which has been shown previously to modify specifically an active site arginine in D-amino acid oxidase (Ferti, C., Curti, B., Simonetta, M. P., Ronchi, S., Galliano, M., and Minchiotti, L. (1981) Eur. J. Biochem. 119, 553-557) destroys the ability of D-amino acid oxidase to stabilize the benzoquinoid type spectrum of 8-mercapto-FAD and destroys the ability to form a flavin N-5 adduct with sulfite. Both of these properties have been attributed to the presence of such a group. The active site lysine, histidine, and tyrosine have been ruled out as possibilities for such a group. In addition, the reactivity of flavoproteins containing 8-mercaptoflavin with sulfite has been examined and falls into the same two general classes as the reactivity of the native flavoproteins: oxidases form N-5 adducts while all of the other 8-mercaptoflavoproteins examined do not, forming instead the 8-sulfonate flavin.     

A hydrogen peroxide-forming NADH oxidase that functions as an alkyl hydroperoxide reductase in Amphibacillus xylanus

The Amphibacillus xylanus NADH oxidase, which catalyzes the reduction of oxygen to hydrogen peroxide with beta-NADH, can also reduce hydrogen peroxide to water in the presence of free flavin adenine dinucleotide (FAD) or the small disulfide-containing Salmonella enterica AhpC protein. The enzyme has two disulfide bonds, Cys128-Cys131 and Cys337-Cys340, which can act as redox centers in addition to the enzyme-bound FAD (K. Ohnishi, Y. Niimura, M. Hidaka, H. Masaki, H. Suzuki, T. Uozumi, and T. Nishino, J. Biol. Chem. 270:5812-5817, 1995). The NADH-FAD reductase activity was directly dependent on the FAD concentration, with a second-order rate constant of approximately 2.0 x 10(6) M(-1) s(-1). Rapid-reaction studies showed that the reduction of free flavin occurred through enzyme-bound FAD, which was reduced by NADH. The peroxidase activity of NADH oxidase in the presence of FAD resulted from reduction of peroxide by free FADH(2) reduced via enzyme-bound FAD. This peroxidase activity was markedly decreased in the presence of oxygen, since the free FADH(2) is easily oxidized by oxygen, indicating that this enzyme system is unlikely to be functional in aerobic growing cells. The A. xylanus ahpC gene was cloned and overexpressed in Escherichia coli. When the NADH oxidase was coupled with A. xylanus AhpC, the peroxidase activity was not inhibited by oxygen. The V(max) values for hydrogen peroxide and cumene hydroperoxide reduction were both approximately 150 s(-1). The K(m) values for hydrogen peroxide and cumene hydroperoxide were too low to allow accurate determination of their values. Both AhpC and NADH oxidase were induced under aerobic conditions, a clear indication that these proteins are involved in the removal of peroxides under aerobic growing conditions.     

Streptococcus mutans H2O2-forming NADH oxidase is an alkyl hydroperoxide reductase protein

Nox-1 from Streptococcus mutans, the bacteria which cause dental caries, was previously identified as an H2O2-forming reduced nicotinamide adenine dinucleotide (NADH) oxidase. Nox-1 is homologous with the flavoprotein component, AhpF, of Salmonella typhimurium alkyl hydroperoxide reductase. A partial open reading frame upstream of nox1, homologous with the other (peroxidase) component, ahpC, from the S. typhimurium system, was also identified. We report here the complete sequence of S. mutans ahpC. Analyses of purified AhpC together with Nox-1 have verified that these proteins act as a cysteine-based peroxidase system in S. mutans, catalyzing the NADH-dependent reduction of organic hydroperoxides or H2O2 to their respective alcohols and/or H2O. These proteins also catalyze the four-electron reduction of O2 to H2O2, clarifying the role of Nox-1 as a protective protein against oxygen toxicity. Major differences between Nox-1 and AhpF include: (i) the absolute specificity of Nox-1 for NADH; (ii) lower amounts of flavin semiquinone and a more prominent FADH2 to NAD+ charge transfer absorbance band stabilized by Nox-1; and (iii) even higher redox potentials of disulfide centers relative to flavin for Nox-1. Although Nox-1 and AhpC from S. mutans were shown to play a protective role against oxidative stress in vitro and in vivo in Escherichia coli, the lack of a significant effect on deletion of these genes from S. mutans suggests the presence of additional antioxidant proteins in these bacteria.     

8-Mercaptoflavins as active site probes of flavoenzymes.

Representative examples of the various classes of flavoproteins have been converted to their apoprotein forms and the native flavin replaced by 8-mercapto-FMN or 8-mercapto-FAD. The spectral and catalytic properties of the modified enzymes are characteristically different from one group to another; the results suggest that flavin interactions at positions N(1) or N(5) of the flavin chromophore have profound influences on the properties of the flavoprotein. 1. The 8-thiolate anion form of 8-mercaptoflavin has an absorption maximum in the region 520 to 550 nm epsilon approximately 30 mM-1 cm-1). This form is retained on binding to flavoproteins whose physiological reactions involve obligatory one-electron transfers (e.g. flavodoxin, NADPH-cytochrome P-450 reductase). In the native form these enzymes stabilize the blue neutral radical of the flavin. A radical form of 8-mercaptoflavin is also stabilized by these proteins. 2. The p-quinoid form of 8-mercaptoflavin has an absorption maximum in the range 560 to 600 nm (epsilon approximately 30 mM-1 cm-1). This form is stabilized on binding to flavoproteins of the dehydrogenase-oxidase class (e.g. glucose oxidase, D-amino acid oxidase, lactate oxidase, Old Yellow Enzyme). These same enzymes in their native flavin form stabilize the red semiquinone, and have a pronounced reactivity with sulfite to form flavin N(5)-sulfite adducts. These properties of the native enzyme, including the ability to react with nitroalkane carbanions, are not exhibited by the 8-mercaptoflavoproteins. 3. A group of flavoenzymes fails to conform strictly to the above classification, exhibiting some properties of both classes. These include the examples of flavoprotein hydroxylases and transhydrogenases studied. 4. The riboflavin-binding protein of hen egg whites binds 8-mercaptoriboflavin preferentially in the unionized state, resulting in a shift in pK from 3.8 with free 8-mercaptoriboflavin to greater than or equal to 9.0 with the protein-bound form.     

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)     

Kinetic mechanism and quaternary structure of Aminobacter aminovorans NADH:flavin oxidoreductase: an unusual flavin reductase with bound flavin

The homodimeric NADH:flavin oxidoreductase from Aminobacter aminovorans is an NADH-specific flavin reductase herein designated FRD(Aa). FRD(Aa) was characterized with respect to purification yields, thermal stability, isoelectric point, molar absorption coefficient, and effects of phosphate buffer strength and pH on activity. Evidence from this work favors the classification of FRD(Aa) as a flavin cofactor-utilizing class I flavin reductase. The isolated native FRD(Aa) contained about 0.5 bound riboflavin-5'-phosphate (FMN) per enzyme monomer, but one bound flavin cofactor per monomer was obtainable in the presence of excess FMN or riboflavin. In addition, FRD(Aa) holoenzyme also utilized FMN, riboflavin, or FAD as a substrate. Steady-state kinetic results of substrate titrations, dead-end inhibition by AMP and lumichrome, and product inhibition by NAD(+) indicated an ordered sequential mechanism with NADH as the first binding substrate and reduced FMN as the first leaving product. This is contrary to the ping-pong mechanism shown by other class I flavin reductases. The FMN bound to the native FRD(Aa) can be fully reduced by NADH and subsequently reoxidized by oxygen. No NADH binding was detected using 90 microM FRD(Aa) apoenzyme and 300 microM NADH. All results favor the interpretation that the bound FMN was a cofactor rather than a substrate. It is highly unusual that a flavin reductase using a sequential mechanism would require a flavin cofactor to facilitate redox exchange between NADH and a flavin substrate. FRD(Aa) exhibited a monomer-dimer equilibrium with a K(d) of 2.7 microM. Similarities and differences between FRD(Aa) and certain flavin reductases are discussed.     

An improved method for the determination of NADH oxidase in the presence of NADH peroxidase in lactic acid bacteria

The complexity of the coupled NADH oxidase-NADH peroxidase enzyme system in lactic acid bacteria makes it difficult to simultaneously determine the individual levels of both these enzymes spectrophotometrically. This study describes an improved assay to accurately determine low concentrations of NADH oxidase from enzyme suspensions containing NADH oxidase and NADH peroxidase. For the standardisation of the assay, pure NADH oxidase and NADH peroxidase were mixed in various proportions and the percentage recovery was estimated by both the currently available assay as well as by the improved assay reported in this study. The recovery of NADH oxidase using the currently available assay ranged from as low as -200% to as high as +102% as against 90-102% in the improved assay. The recovery percentage of NADH peroxidase ranged from 91% to 112% in both assays. The slopes of NADH oxidation by cell-free extracts of six lactic acid bacteria were also measured by both assays for the estimation of NADH oxidase and NADH peroxidase levels. The improved assay can further distinguish between NADH-H(2)O oxidase and NADH-H(2)O(2) oxidase and was successfully applied to identify the type of NADH oxidase in the lactic acid bacteria tested.     

AhpF and other NADH:peroxiredoxin oxidoreductases, homologues of low Mr thioredoxin reductase.

A group of bacterial flavoproteins related to thioredoxin reductase contain an additional approximately 200-amino-acid domain including a redox-active disulfide center at their N-termini. These flavoproteins, designated NADH:peroxiredoxin oxidoreductases, catalyze the pyridine-nucleotide-dependent reduction of cysteine-based peroxidases (e.g. Salmonella typhimurium AhpC, a member of the peroxiredoxin family) which in turn reduce H2O2 or organic hydroperoxides. These enzymes catalyze rapid electron transfer (kcat > 165 s-1) through one tightly bound FAD and two redox-active disulfide centers, with the N-terminal-most disulfide center acting as a redox mediator between the thioredoxin-reductase-like part of these proteins and the peroxiredoxin substrates. A chimeric protein with the first 207 amino acids of S. typhimurium AhpF attached to the N-terminus of Escherichia coli thioredoxin reductase exhibits very high NADPH:peroxiredoxin oxidoreductase and thioredoxin reductase activities. Catalytic turnover by NADH:peroxiredoxin oxidoreductases may involve major domain rotations, analogous to those proposed for bacterial thioredoxin reductase, and cycling of these enzymes between two electron-reduced (EH2) and four electron-reduced (EH4) redox states.     

Analysis of the kinetic and redox properties of the NADH peroxidase R303M mutant: correlation with the crystal structure

The crystal structure of the flavoprotein NADH peroxidase shows that the Arg303 side chain forms a hydrogen bond with the active-site His10 imidazole and is therefore likely to influence the catalytic mechanism. Dithionite titration of an R303M mutant [E(FAD, Cys42-sulfenic acid)] yields a two-electron reduced intermediate (EH(2)) with enhanced flavin fluorescence and almost no charge-transfer absorbance at pH 7.0; the pK(a) for the nascent Cys42-SH is increased by over 3.5 units in comparison with the wild-type EH(2) pK(a) of Cys42-SOH. The crystal structure of the R303M peroxidase has been refined at 2.45 A resolution. In addition to eliminating the Arg303 interactions with His10 and Glu14, the mutant exhibits a significant change in the conformation of the Cys42-SOH side chain relative to FAD and His10 in particular. These and other results provide a detailed understanding of Arg303 and its role in the structure and mechanism of this unique flavoprotein peroxidase.     

Cholesterol oxidase from Brevibacterium sterolicum. The relationship between covalent flavinylation and redox properties

Brevibacterium sterolicum possesses two forms of cholesterol oxidase, one containing noncovalently bound FAD, the second containing a FAD covalently linked to His(69) of the protein backbone. The functional role of the histidyl-FAD bond in the latter cholesterol oxidase was addressed by studying the properties of the H69A mutant in which the FAD is bound tightly, but not covalently, and by comparison with native enzyme. The mutant retains catalytic activity, but with a turnover rate decreased 35-fold; the isomerization step of the intermediate 3-ketosteroid to the final product is also preserved. Stabilization of the flavin semiquinone and binding of sulfite are markedly decreased, this correlates with a lower midpoint redox potential (-204 mV compared with -101 mV for wild-type). Reconstitution with 8-chloro-FAD led to a holoenzyme form of H69A cholesterol oxidase with a midpoint redox potential of -160 mV. In this enzyme form, flavin semiquinone is newly stabilized, and a 3.5-fold activity increase is observed, this mimicking the thermodynamic effects induced by the covalent flavin linkage. It is concluded that the flavin 8alpha-linkage to a (N1)histidine is a pivotal factor in the modulation of the redox properties of this cholesterol oxidase to increase its oxidative power.     

A New Type-II NADH Dehydrogenase from the Archaeon Acidianus ambivalens: Characterization and in vitro Reconstitution of the Respiratory Chain

A new type-II NADH dehydrogenase (NDH-II) was isolated from the hyperthermoacidophilic archaeon Acidianus ambivalens. This enzyme is a monomer with an apparent molecular mass of 47 kDa, containing a covalently bound flavin, and no iron–sulfur clusters. Upon isolation, NDH-II loses activity, which can, nevertheless, be restored by incubation with phospholipids. Catalytically, it is a proficient NADH:caldariella quinone oxidoreductase (130 mmol NADH oxidized/mg protein^-1/min^-1) but it can also donate electrons to synthetic quinones, strongly suggesting its involvement in the respiratory chain. The apparent K_m for NADH was found to be 6 M, both for the purified and membrane-integrated enzyme, thus showing that detergent solubilization and purification did not affect the substrate binding site. Further, it is the first example of a type-II NADH dehydrogenase that contains the flavin covalently attached, which may be related to the need to stabilize the otherwise labile cofactor in a thermophilic environment. A fully operative minimal version of Acidianus ambivalens respiratory system was successfully reconstituted into artificial liposomes, using three basic components isolated from the organism: the type-II NADH dehydrogenase, caldariella quinone, the organism-specific quinone, and the aa₃ type quinol oxidase. This system, which mimics the in vivo chain, is efficiently energized by NADH, driving oxygen consumption by means of the terminal oxidase.     

Chemical mechanism and rate-limiting steps in the reaction catalyzed by Streptococcus faecalis NADH peroxidase

The pH dependence of the kinetic parameters V, V/KNADH, and V/KH2O2 has been determined for the flavoenzyme NADH peroxidase. Both V/KNADH and V/KH2O2 decrease as groups exhibiting pK's of 9.2 and 9.9, respectively, are deprotonated. The V profile decreases by a factor of 5 as a group exhibiting a pK of 7.2 is deprotonated. Primary deuterium kinetic isotope effects on NADH oxidation are observed on V only, and the magnitude of DV is independent of H2O2 concentration at pH 7.5. DV/KNADH is pH independent and equal to 1.0 between pH 6 and pH 9.5, but DV is pH dependent, decreasing from a value of 7.2 at pH 5.5 to 1.9 at pH 9.5. The shape of the DV versus pH profile parallels that observed in the V profile and yields a similar pK of 6.6 for the group whose deprotonation decreases DV. Solvent kinetic isotope effects obtained with NADH or reduced nicotinamide hypoxanthine dinucleotide as the variable substrate are observed on V only, while equivalent solvent kinetic isotope effects on V and V/K are observed when H2O2 is used as the variable substrate. In all cases linear proton inventories are observed. Primary deuterium kinetic isotope effects on V for NADH oxidation decrease as the solvent isotopic composition is changed from H2O to D2O. These data are consistent with a change in the rate-limiting step from a step in the reductive half-reaction at low pH to a step in the oxidative half-reaction at high pH. Analysis of the multiple kinetic isotope effect data suggests that at high D2O concentrations the rate of a single proton transfer step in the oxidative half-reaction is slowed. These data are used to propose a chemical mechanism involving the pH-dependent protonation of a flavin hydroxide anion, following flavin peroxide bond cleavage.     

NADH oxidase activity of mitochondrial apoptosis-inducing factor

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein, which translocates to the nucleus during apoptosis and causes chromatin condensation and large scale DNA fragmentation. Here we report the biochemical characterization of AIF's redox activity. Natural AIF purified from mitochondria and recombinant AIF purified from bacteria (AIFDelta1-120) exhibit NADH oxidase activity, whereas superoxide anion (O(2)(-)) is formed. AIFDelta1-120 is a monomer of 57 kDa containing 1 mol of noncovalently bound FAD/mol of protein. ApoAIFDelta1-120, which lacks FAD, has no NADH oxidase activity. However, native AIFDelta1-120, apoAIFDelta1-120, and the reconstituted (FAD-containing) holoAIFDelta1-120 protein exhibit a similar apoptosis-inducing potential when microinjected into the cytoplasm of intact cells. Inhibition of the redox function, by external addition of superoxide dismutase or covalent derivatization of FAD with diphenyleneiodonium, failed to affect the apoptogenic function of AIFDelta1-120 assessed on purified nuclei in a cell-free system. Conversely, blockade of the apoptogenic function of AIFDelta1-120 with the thiol reagent para- chloromercuriphenylsulfonic acid did not affect its NADH oxidase activity. Altogether, these data indicate that AIF has a marked oxidoreductase activity which can be dissociated from its apoptosis-inducing function.