Evidence for the existence of a tyrosyl residue in the nicotinamide adenine dinucleotide binding site of chicken liver xanthine dehydrogenase

Xanthine-NAD and NADH-methylene blue oxidoreductase activities of chicken liver xanthine dehydrogenase were inactivated by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA), an active site directed reagent for nucleotide binding sites. The inactivation reaction displayed pseudo-first-order kinetics. A double-reciprocal plot of inactivation velocity vs. 5'-FSBA concentration showed that 5'-FSBA and enzyme formed a complex prior to inactivation. NAD protected the enzyme from inactivation by 5'-FSBA in a competitive fashion. The modified enzyme had the same xanthine-dichlorophenolindophenol and xanthine-O2 oxidoreductase activities as the native enzyme, and on addition of xanthine to the modified enzyme, bleaching of the spectrum occurred in the visible region. The amount of radioactivity incorporated into the enzyme by incubation with [14C]-5'-FSBA was parallel to the loss of xanthine-NAD oxidoreductase activity, and the stoichiometry was 1 mol/mol of enzyme-bound FAD for complete inactivation. These results indicated that 5'-FSBA modified specifically the binding site for NAD of chicken liver xanthine dehydrogenase. The incorporated radioactivity was released slowly from 14C-labeled enzyme by incubation with dithiothreitol with concomitant restoration of catalytic activity. The modified residue responsible for inactivation was identified as a tyrosine.     

Differences in redox and kinetic properties between NAD-dependent and O2-dependent types of rat liver xanthine dehydrogenase

Reductive titrations of a NAD-dependent type (type-D) and an O2-dependent type (type-O) of rat liver xanthine dehydrogenase showed that only the type-D enzyme formed a pronounced stable FAD semiquinone (FADH*). The FAD semiquinone was less stabilized in the presence of NAD. The Vmax value for xanthine-NAD activity of type-D enzyme was close to that for xanthine-O2 activity of type-O enzyme, while the Vmax value for xanthine-O2 activity of type-D enzyme was about one-fourth of that of type-O enzyme. The Km value for O2 of type-D enzyme was about five times as large as that of type-O enzyme. The absorbance spectrum of type-D enzyme during turnover with xanthine and O2 as substrates showed a considerable amount of FADH* formation, but that with xanthine and NAD as substrates showed only a negligible one. Low xanthine-O2 activity of type-D enzyme, as compared with that of type-O enzyme, seems to be explained by the conformational change occurring in conversion from type-O to type-D enzyme, which results in different reactivity of FAD to molecular oxygen and a higher fraction of FADH* during turnover. The binding of NAD may possibly increase the fraction of FADH2, resulting in a Vmax value of xanthine-NAD activity almost as high as that of xanthine-O2 activity of type-O enzyme.     

Oxidation--reduction potentials of turkey liver xanthine dehydrogenase and the origins of oxidase and dehydrogenase behaviour in molybdenum-containing hydroxylases.

Redox potentials for the various centres in the enzyme xanthine dehydrogenase (EC 1.2.1.37) from turkey liver determined by potentiometric titration in the presence of mediator dyes, with low-temperature electron-paramagnetic-resonance spectroscopy. Values at 25 degrees C in pyrophosphate buffer, pH 8.2, are: Mo(VI)/Mo(V)(Rapid),-350 +/- 20mV; Mo(V) (Rapid)/Mo(IV), -362 +/- 20mV; Fe-S Iox./Fe-S Ired., -295 +/- 15mV; Fe-S IIox./Fe-S IIred., -292 +/- 15mV; FAD/FADH,-359+-20mV; FADH/FADH2, -366 +/- 20mV. This value of the FADH/FADH2 potential, which is 130mV lower than the corresponding one for milk xanthine oxidase [Cammack, Barber & Bray (1976) Biochem. J. 157, 469-478], accounts for many of the differences between the two enzymes. When allowance is made for some interference by desulpho enzyme, then differences in the enzymes' behaviour in titration with xanthine [Barber, Bray, Lowe & Coughlan (1976) Biochem. J. 153, 297-307] are accounted for by the potentials. Increases in the molybdenum potentials of the enzymes caused by the binding of uric acid are discussed. Though the potential of uric acid/xanthine (-440mV) is favourable for full reduction of the dehydrogenase, nevertheless, during turnover, for kinetic reasons, only FADH and very little FADH2 is produced from it. Since only FADH2 is expected to react with O2, lack of oxidase activity by the dehydrogenase is explained. Reactivity of the two enzymes with NAD+ as electron acceptor is discussed in relation to the potentials.     

Affinity labeling of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine

Incubation of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA) results in the inactivation of the 3 beta-hydroxysteroid dehydrogenase enzyme activity following pseudo-first-order kinetics. A double-reciprocal plot of 1/kobs versus 1/[5'-FSBA] yields a straight line with a positive y intercept, indicative of reversible binding of the inhibitor prior to an irreversible inactivation reaction. The dissociation constant (Kd) for the initial reversible enzyme-inhibitor complex is estimated at 0.533 mM, with k2 = 0.22 min-1. The irreversible inactivation could be prevented by the presence of NAD+ during the incubation, indicating that 5'-FSBA inactivates the 3 beta-hydroxysteroid dehydrogenase activity by reacting at the NAD+ binding site. Although the enzyme was inactivated by incubation with 5'-FSBA, no incorporation of the inhibitor was found in labeling studies using 5'-[p-(fluorosulfonyl)benzoyl] [14C]adenosine. However, the inactivation of 3 beta-hydroxysteroid dehydrogenase activity caused by incubation with 5'-FSBA could be completely reversed by the addition of dithiothreitol. This indicates the presence of at least two cysteine residues at or in the vicinity of the NAD+ binding site, which may form a disulfide bond catalyzed by the presence of 5'-FSBA. The intramolecular cysteine disulfide bridge was found between the cysteine residues in the peptides 274EWGFCLDSR282 and 18IICLLVEEK26, by comparing the [14C]iodoacetic acid labeling before and after recovering the enzyme activity upon the addition of dithiothreitol.     

Studies on chicken liver xanthine dehydrogenase with reference to the problem of non-equivalence of FAD moieties.

1. Reduction of chicken liver xanthine dehydrogenase (xanthine: NAD+ oxidoreductase, EC 1.2.1.37) by xanthine under anaerobic condition proceeded in two phases. This biphasicity may be due to functional and non-functional enzymes in the enzyme preparation. 2. Cyanolysis of a persulfide group of chicken liver enzyme resulted in an inactivation of the enzyme. The non-functional enzyme in the standard enzyme preparation was found to lack persulfide groups at the active sites. 3. The remaining NADH-Methylene Blue oxidoreductase activity, after KI treatment of the xanthine-reduced enzyme of a high flavin activity ratio, is not at the level of 50% of the initial activity, differing from the report suggesting non-equivalence of FAD chromophores. 4. The findings in the present report indicate that FAD chromophores of chicken liver enzyme are essentially equivalent.     

Reactivity of chicken liver xanthine dehydrogenase containing modified flavins

Native FAD was removed from chicken liver xanthine dehydrogenase (XDH) and replaced with a number of artificial flavins of different redox potential. Dithionite titration of the 2-thio-FAD- or 4-thio-FAD (high potential)-containing enzymes showed that the first center to be reduced was the flavin. With native enzyme, iron-sulfur centers are the first to be reduced. With the low potential flavin, 6-OH-FAD, the enzyme-bound flavin was the last center to be reduced in reductive titration with xanthine. These shifts in the reduction profile support the hypothesis that the distribution of reducing equivalents in multi-center oxidation-reduction enzymes of this type is determined by the relative potentials of the centers. The reaction of molecular oxygen with fully reduced 2-thio-FAD XDH or 4-thio-FAD XDH resulted in 5 electron eq being released in a fast phase and one in a slow phase. Reduction of these enzymes by xanthine was limited at a rate comparable to that for the release of urate from native XDH. Xanthine/O2 turnover with these enzymes (and native XDH) resulted in approximately 40-50% of the xanthine reducing equivalents appearing as superoxide. Steady state turnover experiments involving all modified flavin-containing enzymes, as well as native enzyme, showed that shifting the flavin potential either positive or negative relative to FAD caused a decrease in catalytic activity in the xanthine/NAD reductase reaction. In the case of the xanthine/O2 reductase activity, there is no simple obvious relationship between the activity and the redox potential of the reconstituted flavin.     

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.     

Proton stoichiometry in the reduction of the FAD and disulfide of Escherichia coli thioredoxin reductase. Evidence for a base at the active site

The oxidation-reduction midpoint potentials, Em, of the FAD and active site disulfide couples of Escherichia coli thioredoxin reductase have been determined from pH 5.5 to 8.5. The FAD and disulfide couples have similar Em values and thus a linked equilibrium of four microscopic enzyme oxidation-reduction states exists. The binding of phenylmercuric acetate to one enzyme form could be monitored which allowed solving the four microscopic Em values. The Em values at pH 7.0 and 12 degrees C of the four couples of thioredoxin reductase are: (S)2-enzyme-FAD/FADH2 = -0.243 V, (SH)2-enzyme-FAD/FADH2 = -0.260 V, (FAD)-enzyme-(S)2/(SH)2 = -0.254 V, and (FADH2)-enzyme-(S)2/(SH)2 = -0.271 V. Thus, at pH 7.0, the FAD and disulfide moieties have a 0.017-V negative interaction and Em values which are different by 0.011 V. The delta Em/delta pH of the FAD couples E2m and E3m are about 0.060 V/pH throughout the pH range studied, showing an approximately 2-proton stoichiometry of reduction of the enzyme FAD. The delta Em/delta pH of the disulfide couples E1m and E4m are about 0.052 V/pH from pH 5.5 to 8.5, showing an apparently nonintegral proton stoichiometry of reduction of 1.8 in this pH range. This proton stoichiometry suggests the presence of a base with an ionization behavior that is linked to the oxidation-reduction state of the disulfide. A novel method is presented for determining the pK values on oxidized and reduced enzyme which agrees with the less accurate classical method. The proton stoichiometry results are consistent with the presence of a thiol-base ion pair in which the pK of the base is elevated from 7.6 in disulfide containing enzyme to greater than 8.5 upon forming an ion pair with a thiol anion of pK 7.0 generated upon reduction of the disulfide. The fluorescence of the FAD in thioredoxin reductase decreases as the pH is lowered with a pK of 7.0, direct evidence for a base near the FAD probably distinct from the base interacting with the dithiol.     

Comparative study of chicken liver xanthine dehydrogenase and bovine liver xanthine oxidase. dehydrogenase activity of xanthine oxidase (author's transl)]

A method to purify bovine liver xanthine oxidase in described, with which samples of 256-fold specific activity with respect to the initial homogenate are obtained. Bovine liver xanthine oxidase and chicken liver xanthine dehydrogenase with oxygen as electron acceptor exhibit similar profile in pKM and log V versus pH plots. With NAD+ as electron acceptor a different profile in the pKM xanthine plot is obtained for chicken liver xanthine dehydrogenase. However three inflection points at the same pH values appear in all plots. Both enzymes are irreversibly inhibited by pCMB and reversibly by N-ethylmaleimide and by iodoacetamide, with competitive and uncompetitive type inhibitions respectively. These results suggest that NAD+ alters the enzymatic action since its binding to the enzyme antecedes the binding of xanthine to the xanthine oxidase molecule, without undergoing itself any modification. 0.15 M DDT of DTE treatment of bovine liver xanthine oxidase gives to the enzyme a permanent activity with NAD+ without modifying its activity with oxygen. The enzyme thus treated produces parallel straight lines in Lineweaver-Burk plots.     

The oxidation-reduction and electrocatalytic properties of CO dehydrogenase from Oligotropha carboxidovorans

CO dehydrogenase (CODH) from the Gram-negative bacterium Oligotropha carboxidovorans is a complex metalloenzyme from the xanthine oxidase family of molybdenum-containing enzymes, bearing a unique binuclear Mo-S-Cu active site in addition to two [2Fe-2S] clusters (FeSI and FeSII) and one equivalent of FAD. CODH catalyzes the oxidation of CO to CO₂ with the concomitant introduction of reducing equivalents into the quinone pool, thus enabling the organism to utilize CO as sole source of both carbon and energy. Using a variety of EPR monitored redox titrations and spectroelectrochemistry, we report the redox potentials of CO dehydrogenase at pH 7.2 namely Mo^VI/V, Mo^V/IV, FeSI^2+/+, FeSII^2+/+, FAD/FADH and FADH/FADH⁻. These potentials are systematically higher than the corresponding potentials seen for other members of the xanthine oxidase family of Mo enzymes, and are in line with CODH utilising the higher potential quinone pool as an electron acceptor instead of pyridine nucleotides. CODH is also active when immobilised on a modified Au working electrode as demonstrated by cyclic voltammetry in the presence of CO.     

Affinity labeling of Escherichia coli DNA polymerase I by 5'-fluorosulfonylbenzoyladenosine. Identification of the domain essential for polymerization and Arg-682 as the site of reactivity

Preincubation of Escherichia coli DNA polymerase I (pol I) with 5'-fluorosulfonylbenzoyladenosine (5'-FSBA) results in an irreversible inactivation of DNA polymerase activity with concomitant covalent binding of 5'-FSBA to enzyme. pol I-associated 3'-5' exonuclease activity, however, remains unaffected. Kinetic studies of inactivation indicate that the degree of inactivation is directly proportional to the concentration of 5'-FSBA and increases linearly with time. The presence of the metal chelate form of dNTP substrates or template primer, but not the template or primer alone, protects the enzyme from inactivation by 5'-FSBA. A complete inactivation of polymerase activity occurs when 2 mol of 5'-FSBA are covalently linked to 1 mol of enzyme, suggesting two sites of modification. Tryptic peptide mapping of 5'-FSBA-treated enzyme revealed the presence of two distinct peptides containing the affinity label, confirming the presence of two reactive sites in the enzyme. However, we find that only one of the two sites is essential for the polymerase activity since, in the presence of substrate dNTP or template primer during preincubation of enzyme with 5'-FSBA, incorporation of the affinity label is reduced by only 1 mol. Peptide analysis of dNTP or template primer-protected enzyme further revealed that a peptide eluting at 35 min from the C-18 matrix was protected from the 5'-FSBA reaction. It is therefore concluded that this peptide contains the domain essential for polymerase activity. Staphylococcus aureus V-8 protease digestion, amino acid composition, and sequence analysis of this peptide revealed this domain to span residues 669 to 687 in the primary amino acid sequence of pol I, and arginine 682 was found to be the site of 5'-FSBA reactivity.     

Electron paramagnetic resonance properties and oxidation-reduction potentials of the molybdenum,flavin, and iron-sulfur centers of chicken liver xanthine dehydrogenase

The oxidation-reduction potentials of the various prosthetic groups in the native and desulfo forms of chicken liver xanthine dehydrogenase, determined by potentiometric titration in 0.05 m potassium phosphate buffer, pH 7.8, are: Mo(VI)/Mo(V) (native), ?357 mV; Mo(VI)/Mo(V) (desulfo), ?397 mV; Mo(V)/Mo(IV) (native), ?337 mV; Mo(V)/Mo(IV) (desulfo), ?433 mV; FAD/FADH · ?345 mV; FADH · FADH₂, ? 377 mV; (Fe/S)I_ox/(Fe/S)I_red, ?280 mV; (Fe/S)II_ox/(Fe/S)II_red, ? 275 mV. Titration at pH 6.8 revealed that the Mo and FAD centers but not the Fe/S centers are in prototropic equilibrium. Spectroscopic studies on the native and deflavinated enzymes show that environment of the flavin in xanthine dehydrogenase differs from that in bovine milk xanthine oxidase.     

Graphical analysis of interactions between oxidation-reduction sites in two site oxidation-reduction proteins

Many enzymes that catalyze electron-transfer reaction contain multiple oxidation-reduction centers (sites). The oxidation-reduction potential of one site as well as the kinetics of electron transfer through this site may be altered by the state of reduction of a neighboring site. Oxidation-reduction site interactions may be mechanistically important and quantitation of site interactions would aid the interpretation of thermodynamic data and possibly kinetic data. A graphical means to detect and quantitate interactions between oxidation-reduction sites from oxidation-reduction equilibrium data (type A + B in equilibrium C + D) is described and has its roots in the Scatchard analysis of ligand binding equilibria (type A + B in equilibrium C). Oxidation-reduction sites often have distinct physical properties allowing the titration behavior of specific sites to be monitored. Equilibrium measurements on specific sites of a two site protein allow a further analysis of the data which can be combined with the oxidation-reduction Scatchard analysis to solve for all four specific site equilibrium constants. Ligand binding systems can usually measure only total site binding and simplifying assumptions of identical sites or noninteracting sites are required to solve for the site specific equilibrium constants. Thus, specific site equilibrium measurements offer a distinct advantage over total site measurements. The principles of the method are illustrated by applying the graphical analysis to the two site protein, thioredoxin reductase, which contains an oxidation-reduction active site disulfide in addition to FAD. The specific site oxidation-reduction midpoint potentials (Em) of the FAD and disulfide couples of thioredoxin reductase at pH 6.0, 12 degrees C, were found to be FAD/FADH2-enzyme-(S)2 = -0.183 V, FAD/FADH2-enzyme-(SH)2 = -0.199 V, (FAD)-enzyme-(S)2/(SH)2 = -0.202 V, and (FADH2)-enzyme-(S)2/(SH)2 = -0.218 V. Hence, at pH 6.0, the FAD and disulfide sites of thioredoxin reductase have Em values that differ by approximately 0.019 V and have a negative interaction of about 0.016 V.     

Affinity labeling of a tyrosine residue in the ATP binding site of the recA protein from Escherichia coli with 5'-p-fluorosulfonylbenzoyladenosine

We have covalently modified the recA protein from Escherichia coli with the adenine nucleotide analog 5'-p-fluorosulfonylbenzoyladenosine (5'-FSBA). The rate at which the protein is modified shows a sigmoidal dependence on the concentration of 5'-FSBA suggesting that binding of the analog is characterized by positive cooperativity. Covalent modification of the protein results in irreversible inactivation of its single-stranded DNA-dependent ATPase activity such that 100% inactivation is achieved when 25% of the enzyme monomers have been modified. Attachment of 5'-FSBA is specific for the ATP-binding site of recA protein as judged by the following criteria: (i) attachment of the affinity label to the protein appears to saturate at 1 mol of 5'-FSBA/mol of protein; (ii) binding of 5'-FSBA to recA protein is inhibited by ATP and competitive inhibitors of its ATP hydrolytic activity, e.g. adenosine-5'-O-(thiotriphosphate), ADP, UTP, and GTP, but not by adenosine; (iii) attachment of 5'-FSBA to the protein occurs at a single site as determined by high pressure liquid chromatography peptide separation. Following trypsin digestion of recA protein that had been covalently modified with [3H]5'-FSBA we isolated a single labeled peptide (T31) containing the exclusive site of 5'-FSBA attachment. A secondary proteolytic digestion was performed on both 5'-FSBA modified T31 and unmodified T31 using Staphylococcus aureus V8 protease, and by comparison of the amino acid compositions of the resulting peptides we identified Tyr-264 as the exclusive site of 5'-FSBA attachment in recA protein.     

Xanthine dehydrogenase from Pseudomonas putida 86: specificity, oxidation-reduction potentials of its redox-active centers, and first EPR characterization

Xanthine dehydrogenase (XDH) from Pseudomonas putida 86, which was induced 65-fold by growth on hypoxanthine, was purified to homogeneity. It catalyzes the oxidation of hypoxanthine, xanthine, purine, and some aromatic aldehydes, using NAD+ as the preferred electron acceptor. In the hypoxanthine:NAD+ assay, the specific activity of purified XDH was 26.7 U (mg protein)(-1). Its activity with ferricyanide and dioxygen was 58% and 4%, respectively, relative to the activity observed with NAD+. XDH from P. putida 86 consists of 91.0 kDa and 46.2 kDa subunits presumably forming an alpha4beta4 structure and contains the same set of redox-active centers as eukaryotic XDHs. After reduction of the enzyme with xanthine, electron paramagnetic resonance (EPR) signals of the neutral FAD semiquinone radical and the Mo(V) rapid signal were observed at 77 K. Resonances from FeSI and FeSII were detected at 15 K. Whereas the observable g factors for FeSII resemble those of other molybdenum hydroxylases, the FeSI center in contrast to most other known FeSI centers has nearly axial symmetry. The EPR features of the redox-active centers of P. putida XDH are very similar to those of eukaryotic XDHs/xanthine oxidases, suggesting that the environment of each center and their functionality are analogous in these enzymes. The midpoint potentials determined for the molybdenum, FeSI and FAD redox couples are close to each other and resemble those of the corresponding centers in eukaryotic XDHs.     

Affinity labeling of a regulatory site of bovine liver glutamate dehydrogenase.

A new adenosine analog, 3'-p-fluorosulfonyl-benzoyladenosine (3'-FSBA), has been synthesized which reacts covalently with bovine liver glutamate dehydrogenase. Native glutamate dehydrogenase is activated by ADP and inhibited by high concentrations of DPNH. Both of these effects are irreversibly decreased upon incubation of the enzyme with the adenosine analog, 3'-p-fluorosulfonyl-benzoyladenosine (3'-FSBA), while the intrinsic enzymatic activity as measured in the absence of regulatory compounds remains unaltered. A plot of the rate constant for modification as a function of the 3'-FSBA concentration is not linear, suggesting that the adenosine derivative binds to the enzyme (Ki equals 1.0 times 10-4 M) prior to the irreversible modification. Protection against modification by 3'-FSBA is provided by ADP and by high concentrations of DPNH, but not by the inhibitor GTP, the substrate alpha-keto glutarate, the coenzyme TPNH, or low concentrations of the coenzyme DPNH. The isolated altered enzyme contains approximately 1 mol of sulfonylbenzoyladenosine per peptide chain, indicating that a single specific regulatory site has reacted with 3'-tfsba. the modified enzyme exhibits normal Michaelis constants for its substrates and is still inhibited by GTP, albeit at a higher concentration, but it is not inhibited by high concentrations of DPNH. Although ADP does not appreciably activate the modified enzyme, it does (as in the case of the native enzyme) overcome the inhibition of the modified enzyme by GTP. These results suggest that ADP can bind to the modified enzyme, but that its ability to activate is affected indirectly by the modification of the adjacent tdpnh inhibitory site. It is proposed that the regulatory sites for ADP and DPNH are partially overlapping and that 3'FSBA functions as a specific affinity label for the DPNH inhibitory site of glutamate dehydrogenase. It is anticipated that 3'-p-fluorosulfonylbenzolyadenosine may act as an affinity label of other dehydrogenases as well as of other classes of enzymes which use adenine nucleotides as substrates or regulators.     

Effect of nicotinamide adenine dinucleotide on the oxidation-reduction potentials of lipoamide dehydrogenase from pig heart

The effect of NAD+ on lipoamide dehydrogenase from pig heart was investigated physicochemically. The observed and theoretical oxidation-reduction mid-point potentials for the oxidized lipoamide dehydrogenase (E)/two-electron-reduced lipoamide dehydrogenase (EH2) couple in the presence on NAD+ were -218 mV and -251 mV, respectively, at pH 6.0. Therefore, unexpectedly the mid-point potential of the enzyme became more positive on NAD+ binding. Decreases in the fluorescence lifetime and intensity and increase in the degree of polarization of enzyme-bound FAD were observed in the presence of NAD+. Fluorescence quenching of bound FAD by NAD+ was released by phenobarbital. The results suggest that NAD+ strengthens the intramolecular dynamic interaction between the isoalloxazine moiety and adenine moiety of bound FAD, and so alters the mid-point potential of the enzyme. These findings indicate that NAD+ acts not only as an acceptor of electrons from EH2, but also as an effector in the flavin-disulfide interaction of EH2.     

Studies by electron-paramagnetic-resonance spectroscopy and stopped-flow spectrophotometry on the mechanism of action of turkey liver xanthine dehydrogenase.

Studies by e.p.r. (electron-paramagnetic-resonance) spectroscopy and by stopped-flow spectrophotometry on turkey liver xanthine dehydrogenase revealed strong similarities to as well as important differences from the Veillonella alcalescens xanthine dehydrogenase and milk xanthine oxidase. The turkey enzyme is contaminated by up to three non-functional forms, giving molybdenum e.p.r. signals designated Resting I, Resting II and Slow. Slow and to a lesser extent Resting I signals are like those from the Veillonella enzyme, whereas Resting II is very like a resting signal described by K. V. Rajagopolan, P. Handler, G. Palmer & H. Beinert (1968) (J. Biol. Chem. 243, 3784-3796) for aldehyde oxidase. Another non-functional form that gives the Inhibited signal is produced on treatment of the enzyme with formaldehyde. Stopped-flow measurements at 450 nm show that, as for the milk enzyme, reduction by xanthine is rate-limiting in enzyme turnover. The active enzyme gives rise to Very Rapid and Rapid molybdenum(V) e.p.r. signals, as well as to an FADH signal. That these signals are almost indistinguishable from those of the milk enzyme, confirms the similarities between the active sites. There are two types of iron-sulphur centres that give signals like those in the milk enzyme, though with slightly different parameters. Quantitative reduction titration of the functional enzyme with xanthine revealed two important differences between the turkey and the milk enzymes. First, the turkey enzyme FADH/FADH2 system has a redox potential sufficiently low that xanthine is incapable of reducing the flavin completely. This finding presumably explains the very low oxidase activity. Secondly, whereas the Fe/S II chromophore in the milk enzyme has a relatively high redox potential, for the turkey enzyme the value of this potential is lower and similar to that of its Fe/S I chromophore.     

The heterogeneity of arginases in rat tissues.

1. The mid-point reduction potentials of the various groups in xanthine oxidase from bovine milk were determined by potentiometric titration with dithionite in the presence of dye mediators, removing samples for quantification of the reduced species by e.p.r. (electron-paramagnetic-resonance) spectroscopy. The values obtained for the functional enzyme in pyrophosphate buffer, pH8.2, are: Fe/S centre I, -343 +/- 15mV; Fe/S II, -303 +/- 15mV; FAD/FADH-; -351 +/- 20mV; FADH/FADH2, -236 +/-mV; Mo(VI)/Mo(V) (Rapid), -355 +/- 20mV; Mo(V) (Rapid)/Mo(IV), -355 +/- 20mV. 2. Behaviour of the functional enzyme is essentially ideal in Tris but less so in pyrophosphate. In Tris, the potential for Mo(VI)/Mo(V) (Rapid) is lowered relative to that in pyrophosphate, but the potential for Fe/S II is raised. The influence of buffer on the potentials was investigated by partial-reduction experiments with six other buffers. 3. Conversion of the enzyme with cyanide into the non-functional form, which gives the Slow molybdenum signal, or alkylation of FAD, has little effect on the mid-point potentials of the other centres. The potentials associated with the Slow signal are: Mo(VI)/Mo(V) (Slow), -440 +/- 25mV; Mo(V) (Slow)/Mo(IV), -480 +/- 25 mV. This signal exhibits very sluggish equilibration with the mediator system. 4. The deviations from ideal behaviour are discussed in terms of possible binding of buffer ions or anti-co-operative interactions amongst the redox centres.     

Determination of the redox potentials and electron transfer properties of the FAD- and FMN-binding domains of the human oxidoreductase NR1.

Human novel reductase 1 (NR1) is an NADPH dependent diflavin oxidoreductase related to cytochrome P450 reductase (CPR). The FAD/NADPH- and FMN-binding domains of NR1 have been expressed and purified and their redox properties studied by stopped-flow and steady-state kinetic methods, and by potentiometry. The midpoint reduction potentials of the oxidized/semiquinone (-315 +/- 5 mV) and semiquinone/dihydroquinone (-365 +/- 15 mV) couples of the FAD/NADPH domain are similar to those for the FAD/NADPH domain of human CPR, but the rate of hydride transfer from NADPH to the FAD/NADPH domain of NR1 is approximately 200-fold slower. Hydride transfer is rate-limiting in steady-state reactions of the FAD/NADPH domain with artificial redox acceptors. Stopped-flow studies indicate that hydride transfer from the FAD/NADPH domain of NR1 to NADP+ is faster than hydride transfer in the physiological direction (NADPH to FAD), consistent with the measured reduction potentials of the FAD couples [midpoint potential for FAD redox couples is -340 mV, cf-320 mV for NAD(P)H]. The midpoint reduction potentials for the flavin couples in the FMN domain are -146 +/- 5 mV (oxidized/semiquinone) and -305 +/- 5 mV (semiquinone/dihydroquinone). The FMN oxidized/semiquinone couple indicates stabilization of the FMN semiquinone, consistent with (a) a need to transfer electrons from the FAD/NADPH domain to the FMN domain, and (b) the thermodynamic properties of the FMN domain in CPR and nitric oxide synthase. Despite overall structural resemblance of NR1 and CPR, our studies reveal thermodynamic similarities but major kinetic differences in the electron transfer reactions catalysed by the flavin-binding domains.