A mutation in subunit I of cytochrome oxidase from Rhodobacter sphaeroides results in an increase in steady-state activity but completely eliminates proton pumping

The heme-copper oxidases convert the free energy liberated in the reduction of O(2) to water into a transmembrane proton electrochemical potential (protonmotive force). One of the essential structural elements of the enzyme is the D-channel, which is thought to be the input pathway, both for protons which go to form H(2)O ("chemical protons") and for protons that get translocated across the lipid membrane ("pumped protons"). The D-channel contains a chain of water molecules extending about 25 A from an aspartic acid (D132 in the Rhodobacter sphaeroides oxidase) near the cytoplasmic ("inside") enzyme surface to a glutamic acid (E286) in the protein interior. Mutations in which either of these acidic residues is replaced by their corresponding amides (D132N or E286Q) result in severe inhibition of enzyme activity. In the current work, an asparagine located in the D-channel has been replaced by the corresponding acid (N139 to D; N98 in bovine enzyme) with dramatic consequences. The N139D mutation not only completely eliminates proton pumping but, at the same time, confers a substantial increase (150-300%) in the steady-state cytochrome oxidase activity. The N139D mutant of the R. sphaeroides oxidase was further characterized by examining the rates of individual steps in the catalytic cycle. Under anaerobic conditions, the rate of reduction of heme a(3) in the fully oxidized enzyme, prior to the reaction with O(2), is identical to that of the wild-type oxidase and is not accelerated. However, the rate of reaction of the fully reduced enzyme with O(2) is accelerated by the N139D mutation, as shown by a more rapid F --> O transition. Whereas the rates of formation and decay of the oxygenated intermediates are altered, the nature of the oxygenated intermediates is not perturbed by the N139D mutation.     

Mechanism for N₂O generation in bacterial nitric oxide reductase: a quantum chemical study

The catalytic mechanism of reduction of NO to N(2)O in the bacterial enzyme nitric oxide reductase has been investigated using hybrid density functional theory and a model of the binuclear center (BNC) based on the newly determined crystal structure. The calculations strongly suggest a so-called cis:b(3) mechanism, while the commonly suggested trans mechanism is found to be energetically unfavorable. The mechanism suggested here involves a stable cis-hyponitrite, and it is shown that from this intermediate one N-O bond can be cleaved without the transfer of a proton or an electron into the binuclear active site, in agreement with experimental observations. The fully oxidized intermediate in the catalytic cycle and the resting form of the enzyme are suggested to have an oxo-bridged BNC with two high-spin ferric irons antiferromagnetically coupled. Both steps of reduction of the BNC after N(2)O formation are found to be pH-dependent, also in agreement with experiment. Finally, it is found that the oxo bridge in the oxidized BNC can react with NO to give nitrite, which explains the experimental observations that the fully oxidized enzyme reacts with NO, and most likely also the observed substrate inhibition at higher NO concentrations.     

Observation of a novel transient ferryl complex with reduced CuB in cytochrome c oxidase

The reaction between mixed-valence (MV) cytochrome c oxidase from beef heart with H2O2 was investigated using the flow-flash technique with a high concentration of H2O2 (1 M) to ensure a fast bimolecular interaction with the enzyme. Under anaerobic conditions the reaction exhibits 3 apparent phases. The first phase (tau congruent with 25 micros) results from the binding of one molecule of H2O2 to reduced heme a3 and the formation of an intermediate which is heme a3 oxoferryl (Fe4+=O2-) with reduced CuB (plus water). During the second phase (tau congruent with 90 micros), the electron transfer from CuB+ to the heme oxoferryl takes place, yielding the oxidized form of cytochrome oxidase (heme a3 Fe3+ and CuB2+, plus hydroxide). During the third phase (tau congruent with 4 ms), an additional molecule of H2O2 binds to the oxidized form of the enzyme and forms compound P, similar to the product observed upon the reaction of the mixed-valence (i.e., two-electron reduced) form of the enzyme with dioxygen. Thus, within about 30 ms the reaction of the mixed-valence form of the enzyme with H2O2 yields the same compound P as does the reaction with dioxygen, as indicated by the final absorbance at 436 nm, which is the same in both cases. This experimental approach allows the investigation of the form of cytochrome c oxidase which has the heme a3 oxoferryl intermediate but with reduced CuB. This state of the enzyme cannot be obtained from the reaction with dioxygen and is potentially useful to address questions concerning the role of the redox state in CuB in the proton pumping mechanism.     

Electron transfer process in cytochrome bd-type ubiquinol oxidase from Escherichia coli revealed by pulse radiolysis

Cytochrome bd is a two-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and binds hemes b558, b595, and d as the redox metal centers. Taking advantage of spectroscopic properties of three hemes which exhibit distinct absorption peaks, we investigated electron transfer within the enzyme by the technique of pulse radiolysis. Reduction of the hemes in the air-oxidized, resting-state enzyme, where heme d exists in mainly an oxygenated form and partially an oxoferryl and a ferric low-spin forms, occurred in two phases. In the faster phase, radiolytically generated N-methylnicotinamide radicals simultaneously reduced the ferric hemes b558 and b595 with a second-order rate constant of 3 x 10(8) M-1 s-1, suggesting that a rapid equilibrium occurs for electron transfer between two b-type hemes long before 10 micros. In the slower phase, an intramolecular electron transfer from heme b to the oxoferryl and the ferric heme d occurred with the first-order rate constant of 4.2-5.6 x 10(2) s-1. In contrast, the oxygenated heme d did not exhibit significant spectral change. Reactions with the fully oxidized and hydrogen peroxide-treated forms demonstrated that the oxidation and/or ligation states of heme d do not affect the heme b reduction. The following intramolecular electron transfer transformed the ferric and oxoferryl forms of heme d to the ferrous and ferric forms, respectively, with the first-order rate constants of 3.4 x 10(3) and 5.9 x 10(2) s-1, respectively.     

Tyrosine 167: the origin of the radical species observed in the reaction of cytochrome c oxidase with hydrogen peroxide in Paracoccus denitrificans

Determination of the three-dimensional structure of cytochrome c oxidase, the terminal enzyme of the respiratory chain, from Paracoccus denitrificans offers the possibility of site-directed mutagenesis studies to investigate the relationship between the structure and the catalytic function of the enzyme. The mechanism of electron-coupled proton transfer is still, however, poorly understood. The P(M) intermediate of the catalytic cycle is an oxoferryl state the generation of which requires one additional electron, which cannot be provided by the two metal centers. It is suggested that the missing electron is donated to this binuclear site by a tyrosine residue that forms a radical species, which can then be detected in both the P(M) and F(*) intermediates of the catalytic cycle. One possibility to produce P(M) and F(*) intermediates artificially in cytochrome c oxidase is the addition of hydrogen peroxide to the fully oxidized enzyme. Using electron paramagnetic resonance (EPR) spectroscopy, we assign a radical species detected in this reaction to a tyrosine residue. To address the question, which tyrosine residue is the origin of the radical species, several tyrosine variants of subunit I are investigated. These variants are characterized by their turnover rates, as well as using EPR and optical spectroscopy. From these experiments, it is concluded that the origin of the radical species appearing in P(M) and F(*) intermediates produced with hydrogen peroxide is tyrosine 167. The significance of this finding for the catalytic function of the enzyme is discussed.     

NO reduction by nitric-oxide reductase from denitrifying bacterium Pseudomonas aeruginosa: characterization of reaction intermediates that appear in the single turnover cycle

Nitric-oxide reductase (NOR) of a denitrifying bacterium catalyzes NO reduction to N(2)O at the binuclear catalytic center consisting of high spin heme b(3) and non-heme Fe(B). The structures of the reaction intermediates in the single turnover of the NO reduction by NOR from Pseudomonas aeruginosa were investigated using optical absorption and EPR spectroscopies combined with an originally designed freeze-quench device. In the EPR spectrum of the sample, in which the fully reduced NOR was mixed with an NO solution and quenched at 0.5 ms after the mixing, two characteristic signals for the ferrous Fe(B)-NO and the penta-coordinated ferrous heme b(3)-NO species were observed. The CO inhibition of its formation indicated that two NO molecules were simultaneously distributed into the two irons of the same binuclear center of the enzyme in this state. The time- and temperature-dependent EPR spectral changes indicated that the species that appeared at 0.5 ms is a transient reaction intermediate prior to the N(2)O formation, in good agreement with the so-called "trans" mechanism. It was also found that the final state of the enzyme in the single turnover cycle is the fully oxidized state, in which the mu-oxo-bridged ligand is absent between the two irons of its binuclear center, unlike the resting form of NOR as isolated. On the basis of these present findings, we propose a newly developed mechanism for the NO reduction reaction conducted by NOR.     

Spectroscopic studies on the photoreaction of choline oxidase, a flavoprotein, with covalently bound flavin

Formation of the anionic flavosemiquinone was observed spectrophotometrically during the anaerobic photo-irradiation of Alcaligenes sp. choline oxidase in the presence of EDTA. Further irradiation slowly converted the semiquinone form into the fully reduced state. The presence of a catalytic amount of riboflavin greatly enhances the photoreduction rate not only to the semiquinone state but also to the fully reduced state. This semiquinone species has low reactivity toward the substrate, choline or betaine aldehyde, as well as toward oxygen. This low reactivity toward oxygen is unique to the semiquinone form of a flavoprotein oxidase. The oxidized enzyme forms a complex with betaine, the product of the enzymatic reaction of choline oxidase. The dissociation constant for this complex was found to be 17 mM by spectroscopic titration. Anaerobic photo-irradiation of the enzyme with a saturating amount of betaine in the absence of EDTA produces, with no detectable semiquinone formation, an absorption spectrum which resembles (but significantly differs from) that of the fully reduced form. This species was found to comprise two flavin species. One of them is rapidly oxidized to the oxidized form by oxygen and is thus assigned as the fully reduced state. The other is converted slowly to the oxidized form upon aerobic standing in the dark. We tentatively assigned this latter species as a C(4a)-adduct. Formaldehyde was detected as a product of this photoreaction. The amount of formaldehyde formed coincided with that of the fully reduced enzyme. On the basis of the results obtained we propose a mechanism of the photoreaction of the enzyme in the presence of betaine where a C(4a)-adduct and the fully reduced enzyme via an N(5)-adduct are formed. Betaine also affects the dithionite reduction. In the dithionite reduction of the oxidized enzyme, the semiquinone species is an intermediate in the conversion of the oxidized to the fully reduced form, while the reduction of the oxidized enzyme-betaine complex with dithionite produces the fully reduced form without any significant formation of the semiquinone species.     

Single-electron photoreduction of the P(M) intermediate of cytochrome c oxidase

The P(M)-->F transition of the catalytic cycle of cytochrome c oxidase from bovine heart was investigated using single-electron photoreduction and monitoring the subsequent events using spectroscopic and electometric techniques. The P(M) state of the oxidase was generated by exposing the oxidized enzyme to CO plus O2. Photoreduction results in rapid electron transfer from heme a to oxoferryl heme a3 with a time constant of about 0.3 ms, as indicated by transients at 605 nm and 580 nm. This rate is approximately 5-fold more rapid than the rate of electron transfer from heme a to heme a3 in the F-->O transition, but is significantly slower than formation of the F state from the P(R) intermediate in the reaction of the fully reduced enzyme with O2 to form state F (70-90 micros). The approximately 0.3 ms P(M)-->F transition is coincident with a rapid photonic phase of transmembrane voltage generation, but a significant part of the voltage associated with the P(M)-->F transition is generated much later, with a time constant of 1.3 ms. In addition, the P(M)-->F transition of the R. sphaeroides oxidase was also measured and also was shown to have two phases of electrogenic proton transfer, with tau values of 0.18 and 0.85 ms.     

Quantitative reevaluation of the redox active sites of crystalline bovine heart cytochrome c oxidase

Approximately 30% of the iron contained in a bovine heart cytochrome c oxidase preparation was removed by crystallization, giving a molecular extinction coefficient 1.25-1.4 times higher than those reported thus far. Six electron equivalents provided by dithionite were required for complete reduction of the crystalline cytochrome c oxidase preparation. The fully reduced enzyme was oxidized with 4 oxidation equivalents provided by molecular oxygen, giving an absorption spectrum slightly, but significantly, different from that of the original fully oxidized form. Four electron equivalents were required for complete reduction of the O(2)-oxidized enzyme. The O(2)-oxidized form, when exposed to excess amounts of O(2), was converted to the original oxidized form which required 6 electrons for complete reduction. A slow reduction of the O(2)-oxidized form without any external reductant added indicates the existence of internal electron donors for heme irons in the enzyme. These results suggest that the 2 extra oxidation equivalents in the original oxidized form, compared with the O(2)-oxidized form, are due to a bound peroxide produced by O(2) and electrons from the internal donors, consistently with a peroxide at the O(2) reduction site in the crystal structure of the enzyme (Yoshikawa, S., Shinzawa-Itoh, K. , Nakashima, R., Yaono, R., Yamashita, E., Inoue, N., Yao, M., Fei, M. J., Peters Libeu, C., Mizushima, T., Yamaguchi, H., Tomizaki, T., and Tsukihara, T. (1998) Science 280, 1723-1729).     

Oxidation of thiamine on reaction with nitrogen dioxide generated by ferric myoglobin and hemoglobin in the presence of nitrite and hydrogen peroxide

It is shown that nitrogen dioxide oxidizes thiamine to thiamine disulfide, thiochrome, and oxodihydrothiochrome (ODTch). The latter is formed during oxidation of thiochrome by nitrogen dioxide. Nitrogen dioxide was produced by incubation of nitrite with horse ferric myoglobin and human hemoglobin in the presence of hydrogen peroxide. After addition of tyrosine or phenol to aqueous solutions containing oxoferryl forms of the hemoproteins, thiamine, and nitrite, the yield of thiochrome greatly increased, whereas the yield of ODTch decreased. In the presence of high concentrations of tyrosine or phenol compounds ODTch was not formed at all. The neutral form of thiamine with the closed thiazole cycle and minor tricyclic form of thiamine do not enter the heme pocket of the protein and do not interact with the oxoferryl heme complex Fe(IV=O) or porphyrin radical. The tricyclic form of thiamine is oxidized to thiochrome by tyrosyl radicals located on the surface of the hemoprotein. The thiol form of thiamine is oxidized to thiamine disulfide by both hemoprotein tyrosyl radicals and oxoferryl heme complexes. Nitrite and also tyrosine, tyramine, and phenol readily penetrate into the heme pocket of the protein and reduce the oxyferryl complex to ferric cation. These reactions yield nitrogen dioxide as well as tyrosyl and phenoxyl radicals of tyrosine molecules and phenol compounds, respectively. Tyrosyl and phenoxyl radicals of low molecular weight compounds oxidize thiamine only to thiochrome and thiamine disulfide. The effect of oxoferryl forms of myoglobin and hemoglobin, nitrogen dioxide, and phenol on thiamine oxidative transformation as well as antioxidant properties of the hydrophobic thiamine metabolites thiochrome and ODTch are discussed.     

Kinetics of the spectral changes during reduction of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi

Two radical signals with different line widths are seen in the Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi by EPR spectroscopy. The first radical is observed in the oxidized enzyme, and is assigned as a neutral flavosemiquinone. The second radical is observed in the reduced enzyme and is assigned to be the anionic form of flavosemiquinone. The time course of Na+-NQR reduction by NADH, as monitored by stopped-flow optical spectroscopy, shows three distinct phases, the spectra of which suggest that they correspond to the reduction of three different flavin species. The first phase is fast both in the presence and absence of sodium, and is assigned to reduction of FAD to FADH2 at the NADH dehydrogenating site. The rates of the other two phases are strongly dependent on sodium concentration, and these phases are attributed to reduction of two covalently bound FMN's. Combination of the optical and EPR data suggests that a neutral FMN flavosemiquinone preexists in the oxidized enzyme, and that it is reduced to the fully reduced flavin by NADH. The other FMN moiety is initially oxidized, and is reduced to the anionic flavosemiquinone. One-electron transitions of two discrete flavin species are thus assigned as sodium-dependent steps in the catalytic cycle of Na+-NQR.     

Evidence for superoxide generation from the autoxidation of the favism-inducing aglycone divicine

The formation of the superoxide anion radical (O2-) during the autoxidation of divicine, an unstable aglycone involved in the hemolytic anemia occurring in favism, has been demonstrated by EPR with two different procedures. In the first case (chemical method) an O2--mediated reduction of a nitroxide by cysteine was shown to occur when divicine was allowed to cycle between the oxidized and the reduced form. In the second case (enzymatic method) the specific reaction between superoxide and superoxide dismutase was used as superoxide detector. It was shown that the enzyme attained a steady-state condition when mixed with divicine in the presence of air, as monitored by EPR evaluation of the oxidation state of the catalytic copper: this result is a direct, specific indicator of an O2- flux.     

Direct detection of Fe(IV)[double bond]O intermediates in the cytochrome aa3 oxidase from Paracoccus denitrificans/H2O2 reaction

We report the first evidence for the formation of the "607- and 580-nm forms" in the cytochrome oxidase aa3/H2O2 reaction without the involvement of tyrosine 280. The pKa of the 607-580-nm transition is 7.5. The 607-nm form is also formed in the mixed valence cytochrome oxidase/O2 reaction in the absence of tyrosine 280. Steady-state resonance Raman characterization of the reaction products of both the wild-type and Y280H cytochrome aa3 from Paracoccus denitrificans indicate the formation of six-coordinate low spin species, and do not support, in contrast to previous reports, the formation of a porphyrin pi-cation radical. We observe three oxygen isotope-sensitive Raman bands in the oxidized wild-type aa3/H2O2 reaction at 804, 790, and 358 cm-1. The former two are assigned to the Fe(IV)[double bond]O stretching mode of the 607- and 580-nm forms, respectively. The 14 cm-1 frequency difference between the oxoferryl species is attributed to variations in the basicity of the proximal to heme a3 His-411, induced by the oxoferryl conformations of the heme a3-CuB pocket during the 607-580-nm transition. We suggest that the 804-790 cm-1 oxoferryl transition triggers distal conformational changes that are subsequently communicated to the proximal His-411 heme a3 site. The 358 cm-1 mode has been found for the first time to accumulate with the 804 cm-1 mode in the peroxide reaction. These results indicate that the mechanism of oxygen reduction must be reexamined.     

Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol

Phanerochaete chrysosporium decolorized several polyaromatic azo dyes in ligninolytic culture. The oxidation rates of individual dyes depended on their structures. Veratryl alcohol stimulated azo dye oxidation by pure lignin peroxidase (ligninase, LiP) in vitro. Accumulation of compound II of lignin peroxidase, an oxidized form of the enzyme, was observed after short incubations with these azo substrates. When veratryl alcohol was also present, only the native form of lignin peroxidase was observed. Azo dyes acted as inhibitors of veratryl alcohol oxidation. After an azo dye had been degraded, the oxidation rates of veratryl alcohol recovered, confirming that these two compounds competed for ligninase during the catalytic cycle. Veratryl alcohol acts as a third substrate (with H2O2 and the azo dye) in the lignin peroxidase cycle during oxidations of azo dyes.     

Electron paramagnetic resonance observations on the cytochrome c-containing nitrous oxide reductase from Wolinella succinogenes.

Nitrous oxide reductase from Wolinella succinogenes, an enzyme containing one heme c and four Cu atoms/subunit of Mr = 88,000, was studied by electron paramagnetic resonance (EPR) at 9.2 GHz from 6 to 80 K. In the oxidized state, low spin ferric cytochrome c was observed with gz = 3.10 and an axial Cu resonance was observed with g parallel = 2.17 and g perpendicular = 2.035. No signals were detected at g values greater than 3.10. For the Cu resonance, six hyperfine lines each were observed in the g parallel and g perpendicular regions with average separations of 45.2 and 26.2 gauss, respectively. The hyperfine components are attributed to Cu(I)-Cu(II) S = 1/2 (half-met) centers. Reduction of the enzyme with dithionite caused signals attributable to heme c and Cu to disappear; exposure of that sample to N2O for a few min caused the reappearance of the g = 3.10 component and a new Cu signal with g parallel = 2.17 and g perpendicular = 2.055 that lacked the simple hyperfine components attributed to a single species of half-met center. The enzyme lost no activity as the result of this cycle of reduction and reoxidation. EPR provided no evidence for a Cu-heme interaction. The EPR detectable Cu in the oxidized and reoxidized forms of the enzyme comprised about 23 and 20% of the total Cu, respectively, or about one spin/subunit. The enzyme offers the first example of a nitrous oxide reductase which can have two states of high activity that present very different EPR spectra of Cu. These two states may represent enzyme in two different stages of the catalytic cycle.     

pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of cytochrome c oxidase. The role of H2O produced at the oxygen-reduction site

A study is presented on the pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of purified cytochrome c oxidase (COX) from beef heart reconstituted in phospholipid vesicles (COV). Protons were shown to be released from COV both in the oxidative and reductive phases. In the oxidation by O2 of the fully reduced oxidase, the H+/COX ratio for proton release from COV (R --> O transition) decreased from approximately 2.4 at pH 6.5 to approximately 1.8 at pH 8.5. In the direct reduction of the fully oxidized enzyme (O --> R transition), the H+/COX ratio for proton release from COV increased from approximately 0.3 at pH 6.5 to approximately 1.6 at pH 8.5. Anaerobic oxidation by ferricyanide of the fully reduced oxidase, reconstituted in COV or in the soluble case, resulted in H+ release which exhibited, in both cases, an H+/COX ratio of 1.7-1.9 in the pH range 6.5-8.5. This H+ release associated with ferricyanide oxidation of the oxidase, in the absence of oxygen, originates evidently from deprotonation of acidic groups in the enzyme cooperatively linked to the redox state of the metal centers (redox Bohr protons). The additional H+ release (O2 versus ferricyanide oxidation) approaching 1 H+/COX at pH < or = 6.5 is associated with the reduction of O2 by the reduced metal centers. At pH > or = 8.5, this additional proton release takes place in the reductive phase of the catalytic cycle of the oxidase. The H+/COX ratio for proton release from COV in the overall catalytic cycle, oxidation by O2 of the fully reduced oxidase directly followed by re-reduction (R --> O --> R transition), exhibited a bell-shaped pH dependence approaching 4 at pH 7.2. A mechanism for the involvement in the proton pump of the oxidase of H+/e- cooperative coupling at the metal centers (redox Bohr effects) and protonmotive steps of reduction of O2 to H2O is presented.     

Reactions of nitric oxide with tree and fungal laccase

The reactions of nitric oxide (NO) with the oxidized and reduced forms of fungal and tree laccase, as well as with tree laccase depleted in type 2 copper, are reported. The products of the reactions were determined by NMR and mass spectroscopy, whereas the oxidation states of the enzymes were monitored by EPR and optical spectroscopy. All three copper sites in fungal laccase are reduced by NO. In addition, NO forms a specific complex with the reduced type 2 copper. NO similarly reduces all of the copper sites in tree laccase, but it also oxidizes the reduced sites produced by ascorbate or NO reduction. A catalytic cycle is set up in which N2O, NO2-, and various forms of the enzyme are produced. On freezing of fully reduced tree laccase in the presence of NO, the type 1 copper becomes reoxidized. This reaction does not occur with the enzyme depleted in type 2 copper, suggesting that it involves intramolecular electron transfer from the type 1 copper to NO bound to the type 2 copper. When the half-oxidized tree laccase is formed in the presence of NO, a population of molecules exists which exhibits a type 3 EPR signal. A triplet EPR signal is also seen in the same preparation and is attributed to a population of the enzyme molecules in which NO is bound to the reduced copper of a half-oxidized type 3 copper site.     

Thiamine inhibits formation of dityrosine,a specific marker of oxidative injury,in reactions catalyzed by oxoferryl forms of hemoglobin

Effects of thiamine and its derivatives on inhibition of dityrosine formation were studied in reactions catalyzed by oxoferryl forms of hemoglobin. At high thiamine concentrations, a complete inhibition of dityrosine formation was observed due to interaction of tyrosyl radicals with thiamine tricyclic and thiol forms. In neutral and alkaline media, tyrosyl radicals oxidized thiamine to thiochrome, oxodihydrothiochrome, and thiamine disulfide. In the absence of tyrosine, oxoferryl forms of hemoglobin manifested peroxidase activity towards thiamine and its phosphate esters by inducing their oxidation to disulfide compounds, thiochrome, oxodihydrothiochrome, and their phosphate esters, respectively, in neutral media. Thiamine and its phosphate esters were oxidized by both oxoferryl forms of hemoglobin, viz., +*Hb(IV=O) (compound I with an additional radical on the globin) and Hb(IV=O) (compound II). Putative mechanisms of thiamine conversions under oxidative stress and the protective role of hydrophobic thiamine metabolites are discussed.     

Examination of the reaction of fully reduced cytochrome oxidase with hydrogen peroxide by flow-flash spectroscopy

The reaction of cytochrome c oxidase with hydrogen peroxide has been of great value in generating and characterizing oxygenated species of the enzyme that are identical or similar to those formed during turnover of the enzyme with dioxygen. Most previous studies have utilized relatively low peroxide concentrations (millimolar range). In the current work, these studies have been extended to the examination of the kinetics of the single turnover of the fully reduced enzyme using much higher concentrations of peroxide to avoid limitations by the bimolecular reaction. The flow-flash method is used, in which laser photolysis of the CO adduct of the fully reduced enzyme initiates the reaction following rapid mixing of the enzyme with peroxide, and the reaction is monitored by observing the absorbance changes due to the heme components of the enzyme. The following reaction sequence is deduced from the data. (1) The initial product of the reaction appears to be heme a(3) oxoferryl (Fe(4+)=O(2)(-) + H(2)O). Since the conversion of ferrous to ferryl heme a(3) (Fe(2+) to Fe(4+)) is sufficient for this reaction, presumably Cu(B) remains reduced in the product, along with Cu(A) and heme a. (2) The second phase of the reaction is an internal rearrangement of electrons and protons in which the heme a(3) oxoferryl is reduced to ferric hydroxide (Fe(3+)OH(-)). In about 40% of the population, the electron comes from heme a, and in the remaining 60% of the population, Cu(B) is oxidized. This step has a time constant of about 65 micros. (3) The third apparent phase of the reaction includes two parallel reactions. The population of the enzyme with an electron in the binuclear center reacts with a second molecule of peroxide, forming compound F. The population of the enzyme with the two electrons on heme a and Cu(A) must first transfer an electron to the binuclear center, followed by reaction with a second molecule of peroxide, also yielding compound F. In each of these reaction pathways, the reaction time is 100-200 micros, i.e., much faster than the rate of reaction of peroxide with the fully oxidized enzyme. Thus, hydrogen peroxide is an efficient trap for a single electron in the binuclear center. (4) Compound F is then reduced by the final available electron, again from heme a, at the same rate as observed for the reduction of compound F formed during the reaction of the fully reduced oxidase with dioxygen. The product is the fully oxidized enzyme (heme a(3) Fe(3+)OH(-)), which reacts with a third molecule of hydrogen peroxide, forming compound P. The rate of this final reaction step saturates at high concentrations of peroxide (V(max) = 250 s(-)(1), K(m) = 350 mM). The data indicate a reaction mechanism for the steady-state peroxidase activity of the enzyme which, at pH 7.5, proceeds via the single-electron reduction of the binuclear center followed by reaction with peroxide to form compound F directly, without forming compound P. Peroxide is an efficient trap for the one-electron-reduced state of the binuclear center. The results also suggest that the reaction of hydrogen peroxide to the fully oxidized enzyme may be limited by the presence of hydroxide associated with the heme a(3) ferric species. The reaction of hydrogen peroxide with heme a(3) is very substantially accelerated by the availability of an electron on heme a, which is presumably transferred to the binuclear center concomitant with a proton that can convert the hydroxide to water, which is readily displaced.     

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.