Oxidase Turnover Research Articles

Reaction of the carbonate radical with the spin-trap 5,5-dimethyl-1-pyrroline- N-oxide in chemical and cellular systems: Pulse radiolysis, electron paramagnetic resonance, and kinetic-competition studies

Carbonate radicals (CO 3 − ) can be formed biologically by the reaction of OH with bicarbonate, the decomposition of the peroxynitrite–carbon dioxide adduct (ONOOCO 2 −), and enzymatic activities, i.e., peroxidase activity of CuZnSOD and xanthine oxidase turnover in the presence of bicarbonate. It has been reported that the spin-trap DMPO reacts with CO 3 − to yield transient species to yield finally the DMPO–OH spin adduct. In this study, the kinetics of reaction of CO 3 − with DMPO were studied by pulse radiolysis, yielding a second-order rate constant of 2.5 × 10 6 M − 1 s − 1 . A Fenton system, composed of Fe II–DTPA plus H 2O 2, generated OH that was trapped by DMPO; the presence of 50–500 mM bicarbonate, expected to convert OH to CO 3 − , markedly inhibited DMPO–OH formation. This was demonstrated to be due mainly to a fast reaction of CO 3 − with Fe II–DTPA ( k = 6.1 × 10 8 M − 1 s − 1 ), supported by kinetic analysis. Generation of CO 3 − by the Fenton system was further proved by analysis of tyrosine oxidation products: the presence of bicarbonate caused a dose-dependent inhibition of 3,4-dihydroxiphenylalanine with a concomitant increase of 3,3′-dityrosine yields, and the presence of DMPO inhibited tyrosine oxidation, in agreement with the rate constants with OH or CO 3 − . Similarly, the formation of CO 3 − by CuZnSOD/H 2O 2/bicarbonate and peroxynitrite–carbon dioxide was supported by DMPO hydroxylation and kinetic competition data. Finally, the reaction of CO 3 − with DMPO to yield DMPO–OH was shown in peroxynitrite-forming macrophages. In conclusion, CO 3 − reacts quite rapidly with DMPO and may contribute to DMPO–OH yields in chemical and cellular systems; in turn, the extent of oxidation of other target molecules (such as tyrosine) by CO 3 − will be sensitive to the presence of DMPO.

A Flavo-Diiron Protein from Desulfovibrio vulgaris with Oxidase and Nitric Oxide Reductase Activities. Evidence for an in Vivo Nitric Oxide Scavenging Function

A few members of a widespread class of bacterial and archaeal flavo-diiron proteins, dubbed FprAs, have been shown to function as either oxidases (dioxygen reductases) or scavenging nitric oxide reductases, but the questions of which of these functions dominates in vivo for a given FprA and whether all FprAs function as oxidases or nitric oxide reductases remain to be clarified. To address these questions, an FprA has been characterized from the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris. The gene encoding this D. vulgaris FprA lies downstream of an operon encoding superoxide reductase and rubredoxin, consistent with an O(2)-scavenging oxidase function for this FprA. The recombinant D. vulgaris FprA can indeed serve as the terminal component of an NADH oxidase. However, this oxidase turnover results in irreversible inactivation of the enzyme. On the other hand, the recombinant D. vulgaris FprA shows robust anaerobic nitric oxide reductase activity in vitro and also protects a nitric oxide-sensitive Escherichia coli strain against exposure to exogenous nitric oxide. It is, therefore, proposed that this D. vulgaris FprA functions as a scavenging nitric oxide reductase in vivo and that this activity protects D. vulgaris against anaerobic exposure to nitric oxide. The location of a gene encoding a second FprA homologue in the D. vulgaris genome also suggests its involvement in nitrogen oxide metabolism.

Production of the Carbonate Radical Anion during Xanthine Oxidase Turnover in the Presence of Bicarbonate

Xanthine oxidase is generally recognized as a key enzyme in purine catabolism, but its structural complexity, low substrate specificity, and specialized tissue distribution suggest other functions that remain to be fully identified. The potential of xanthine oxidase to generate superoxide radical anion, hydrogen peroxide, and peroxynitrite has been extensively explored in pathophysiological contexts. Here we demonstrate that xanthine oxidase turnover at physiological pH produces a strong one-electron oxidant, the carbonate radical anion. The radical was shown to be produced from acetaldehyde oxidation by xanthine oxidase in the presence of catalase and bicarbonate on the basis of several lines of evidence such as oxidation of both dihydrorhodamine 123 and 5,5-dimethyl-1-pyrroline-N-oxide and chemiluminescence and isotope labeling/mass spectrometry studies. In the case of xanthine oxidase acting upon xanthine and hypoxanthine as substrates, carbonate radical anion production was also evidenced by the oxidation of 5,5-dimethyl-1-pyrroline-N-oxide and of dihydrorhodamine 123 in the presence of uricase. The results indicated that Fenton chemistry occurring in the bulk solution is not necessary for carbonate radical anion production. Under the conditions employed, the radical was likely to be produced at the enzyme active site by reduction of a peroxymonocarbonate intermediate whose formation and reduction is facilitated by the many xanthine oxidase redox centers. In addition to indicating that the carbonate radical anion may be an important mediator of the pathophysiological effects of xanthine oxidase, the results emphasize the potential of the bicarbonate-carbon dioxide pair as a source of biological oxidants.

Stereoselective Hydrogen Abstraction by Galactose Oxidase

The fungal enzyme galactose oxidase is a radical copper oxidase that catalyzes the oxidation of a broad range of primary alcohols to aldehydes. Previous mechanistic studies have revealed a large substrate deuterium kinetic isotope effect on galactose oxidase turnover whose magnitude varies systematically over a series of substituted benzyl alcohols, reflecting a change in the character of the transition state for substrate oxidation. In this work, these detailed mechanistic studies have been extended using a series of stereospecifically monodeuterated substrates, including 1-O-methyl-alpha-D-galactose as well as unsubstituted benzyl alcohol and 3- and 4-methoxy and 4-nitrobenzyl derivatives. Synthesis of all of these substrates was based on oxidation of the alpha,alpha'-dideuterated alcohol to the corresponding (2)H-labeled aldehyde, followed by asymmetric hydroboration using alpha-pinene/9-BBN reagents to form the stereoisomeric alcohols. Products from enzymatic oxidation of each of these substrates were characterized by mass spectrometry to quantitatively evaluate the substrate dependence of the stereoselectivity of the catalytic reaction. For all of these substrates, the selectivity for pro-S hydrogen abstraction was at least 95%. This selectivity appears to be a direct consequence of constraints imposed by the enzyme on the orientation of substrates bearing a branched beta-carbon. Steady state analysis of kinetic isotope effects on V/K has resolved individual contributions from primary and alpha-secondary kinetic isotope effects in the reaction, providing a test for the involvement of an electron transfer redox equilibrium in the oxidation process. Multiple isotope effect measurements utilizing simultaneous labeling of the substrate and solvent have contributed to refinement of the relation between proton transfer and hydrogen atom transfer steps in substrate oxidation.

Changing the Conformation State of Cytochrome b558 Initiates NADPH Oxidase Activation: MRP8/MRP14 REGULATION

Phagocyte NADPH oxidase generates O2. for defense mechanisms and cellular signaling. Myeloid-related proteins MRP8 and MRP14 of the S100 family are EF-hand calcium-binding proteins. MRP8 and MRP14 were co-isolated from neutrophils on an anti-p47phox matrix with oxidase cytosolic factors and identified by mass spectrometry. MRP8 and MRP14 are absent from Epstein-Barr virus-immortalized B lymphocytes, and, coincidentally, these cells display weak oxidase activity compared with neutrophils. MRP8/MRP14 that was purified from neutrophils enhanced oxidase turnover of B cells in vitro, suggesting that MRP8/MRP14 is involved in the activation process. This was confirmed ex vivo by co-transfection of Epstein-Barr virus-transformed B lymphocytes with genes encoding MRP8 and MRP14. In a semi-recombinant cell-free assay, recombinant MRP8/MRP14 increased the affinity of p67phox for cytochrome b558 synergistically with p47phox. Moreover, MRP8/MRP14 initiated oxidase activation on its own, through a calcium-dependent specific interaction with cytochrome b558 as shown by atomic force microscopy and a structure-function relationship investigation. The data suggest that the change of conformation in cytochrome b558, which initiates the electron transfer, can be mediated by effectors other than oxidase cytosolic factors p67phox and p47phox. Moreover, MRP8/MRP14 dimer behaves as a positive mediator of phagocyte NADPH oxidase regulation.

A role for subunit III in proton uptake into the D pathway and a possible proton exit pathway in Rhodobacter sphaeroides cytochrome c oxidase.

Protons are transferred from the inner surface of cytochrome c oxidase to the active site by the D and K pathways, as well as from the D pathway to the outer surface by a largely undefined proton exit route. Alteration of the initial proton acceptor of the D pathway, D132, to alanine has previously been shown to greatly inhibit oxidase turnover and slow proton uptake into the D pathway. Here it is shown that the removal of subunit III restores a substantial rate of O(2) reduction to D132A. Presumably an alternative proton acceptor for the D pathway becomes active in the absence of subunit III and D132. Thus, in the absence of subunit III cytochrome oxidase shows greater flexibility in terms of proton entry into the D pathway. In the presence of DeltaPsi and DeltapH, turnover of the wild-type oxidase or D132A is slower in the absence of subunit III. Comparison of the turnover rates of subunit III-depleted wild-type oxidase to those of the zinc-inhibited wild-type oxidase containing subunit III, both reconstituted into vesicles, leads to the hypothesis that the absence of subunit III inhibits the ability of the normal proton exit pathway to take up protons from the outside in the presence of DeltaPsi and DeltapH. Thus, subunit III appears to affect the transfer of protons from both the inner and outer surfaces of cytochrome oxidase, perhaps accounting for the long-observed lower efficiency of proton pumping by the subunit III-depleted oxidase.

Substitutions for glutamate 101 in subunit II of cytochrome c oxidase from Rhodobacter sphaeroides result in blocking the proton-conducting K-channel.

Two functional input pathways for protons have been characterized in the heme-copper oxidases: the D-channel and the K-channel. These two proton-conducting channels have different functional roles and have been defined both by X-ray crystallography and by the characterization of site-directed mutants. Whereas the entrance of the D-channel is well-defined as D132(I) (subunit I; Rhodobacter sphaeroides numbering), the entrance of the K-channel has not been clearly defined. Previous mutagenesis studies of the cytochrome bo(3) quinol oxidase from Escherichia coli implicated an almost fully conserved glutamic acid residue within subunit II as a likely candidate for the entrance of the K-channel. The current work examines the properties of mutants of this conserved glutamate in the oxidase from R. sphaeroides (E101(II)I,A,C,Q,D,N,H) and residues in the immediate vicinity of E101(II). It is shown that virtually any substitution for E101(II), including E101(II)D, strongly reduces oxidase turnover (to 8-29%). Furthermore, the low steady-state activity correlates with an inhibition of the rate of reduction of heme a(3) prior to the reaction with O(2). These are phenotypes expected of K-channel mutants. It is concluded that the predominant entry point for protons going into the K-channel of cytochrome oxidase is the surface-exposed glutamic acid E101(II) in subunit II.

Biochemical and immunochemical properties of B lymphocyte cytochrome b558

Like neutrophils, Epstein–Barr virus (EBV)-immortalized B lymphocytes express all constituents of the NADPH oxidase complex necessary to generate superoxide anion O 2 −. The NADPH oxidase activity in EBV-B lymphocytes is only 5% of that measured in neutrophils upon PMA stimulation. Cytochrome b 558 is the sole redox membrane component of NADPH oxidase; it is the protein core around which cytosolic factors assemble in order to mediate oxidase activity. In the present study, we have compared the structural and functional properties of cytochrome b 558 from EBV-B lymphocytes and neutrophils. Cytochrome b 558 from EBV-B lymphocyte plasma membrane, like that from neutrophils, is characterized by a heterodimeric structure with a highly glycosylated β subunit, known as gp91-phox. While the amount of cytochrome b 558 recovered after purification from EBV-B lymphocytes (∼0.24 nmol from 10 10 cells) was low compared to that recovered from neutrophils (∼10 nmol), the biochemical properties of purified cytochrome b 558 from both EBV-B lymphocytes and neutrophils were quite similar with respect to their differential spectra, redox potential, and FAD binding site. Once cytochrome b 558 was extracted from the EBV-B lymphocyte membrane, it was able to mediate, in a reconstituted system of O 2 − production the same oxidase turnover as that found for cytochrome b 558 extracted from neutrophils. A comparison between membrane bound and soluble cytochrome b 558 suggested that the weak oxidase activity measured in intact EBV-B cells might be the result not only of the small amount of expressed cytochrome b 558, but also of a defect of the activation process in lymphocyte membrane.

Fatty acids as modulators of cytochrome c oxidase in proteoliposomes.

The control of cytochrome c oxidase turnover in proteoliposomes by membrane potential (delta psi) and by pH gradient (delta pH) is probably kinetic in nature, and inhibition by valinomycin and stimulation by nigericin indicate that delta pH exerts a greater influence than does an equivalent delta psi. Oleic acid at 100 microM removes all delta psi and delta pH control, whereas a similar concentration of palmitic acid increases turnover but does not completely abolish control. Valinomycin acts synergistically with both fatty acids, indicating that the latter can act as H+/K+ exchangers, but neither fatty acid alone markedly affects delta pH, showing that they cannot fully mimic nigericin. Oleate, but not palmitate, diminishes delta psi, and can move electrophoretically as oleate anion. Submicromolar palmitic acid concentrations partly stimulate turnover in delta psi- and delta pH-controlled proteoliposomes, as reported by Labonia, Muller and Azzi [(1988) Biochem. J. 254, 130-145], which might represent a direct effect on cytochrome c oxidase. The ubiquity of fatty acids in biological membranes suggests that these substances might be responsible for limiting respiratory control and enzyme activity in vivo.

Intramolecular electron transfer in the oxidation of amines by methylamine oxidase from Arthrobacter P1

The intramolecular electron-transfer rate constant for the Cu(II)–topaNH2⇌ Cu(I)–topaSQ equilibrium in methylamine oxidase has been measured by temperature-jump relaxation techniques. At pH 7.0 the estimated kobs = 150±30 s–1 for both methylamine and benzylamine; assuming the equilibrium constant is ≈0.7–1 at pH 7.0 and 296 K, this would correspond to a forward electron-transfer rate constant kET≈ 60–75 s–1. Although substantially slower than the previously determined kET≈ 20 000 s–1 for pea seedling amine oxidase [5] steady-state kinetics measurements established that kET > kcat≈ 4–10 s–1. Thus the Cu(I)-semiquinone state is a viable intermediate in methylamine oxidase turnover.

Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase.

Diphenyleneiodonium (DPI) and its analogues have been previously shown to react via a radical mechanism whereby an electron is abstracted from a nucleophile to form a radical, which then adds back to the nucleophile to form covalent adducts [Banks (1966) Chem. Rev. 66, 243-266]. We propose that the inhibition of neutrophil NADPH oxidase by DPI occurs via a similar mechanism. A reduced redox centre in the oxidase could serve as electron donor to DPI, and inhibition would occur after direct phenylation of the redox cofactor, or of adjacent amino acid groups by the DPI radical. In the absence of an activatory stimulus, human neutrophil NADPH-oxidase was not inhibited by DPI. The Ki for time-dependent inhibition by DPI of human neutrophil membrane NADPH oxidase was found to be 5.6 microM. Inhibitory potency of DPI was shown to be directly related to rate of enzyme turnover, indicating the need for a reduced redox centre. Adducts were formed between photoreduced flavin (FAD or FMN) and inhibitor (DPI or diphenyliodonium). These were separated by h.p.l.c. and characterized by absorbance spectroscopy, 1H-n.m.r. and fast-atom-bombardment m.s. and found to have properties consistent with substituted 4a,5-dihydroflavins. After incubation of pig neutrophil membranes with DPI, the quantity of recoverable intact flavin was greatly diminished when NADPH was present to initiate oxidase turnover, indicating that the flavin may be the site of DPI activation. These results may provide a common mechanism of action for iodonium compounds as inhibitors of other flavoenzymes.

Control of proteoliposomal cytochrome c oxidase: the partial reactions

The steady-state spectroscopic behaviour and the turnover of cytochrome c oxidase incorporated into proteoliposomes have been investigated as functions of membrane potential and pH gradient. The respiration rate is almost linearly dependent on [cytochrome c2+] at high flux, but while the cytochrome a redox state is always dependent on the [cytochrome c2+] steady state, it reaches a maximum reduction level less than 100% in each case. The maximal aerobic steady-state reduction level of cytochrome a is highest in the presence of valinomycin and lowest in the presence of nigericin. The proportion of [cytochrome c2+] required to achieve 50% of maximal reduction of cytochrome a varies with the added ionophores; the apparent redox potential of cytochrome a is most positive in the fully decontrolled system (plus valinomycin and nigericin). At low levels of cytochrome a reduction, the rate of respiration is no longer a linear function of [cytochrome c2+], but is dependent upon the redox state of both cytochromes a and c. That is, proteoliposomal oxidase does not follow Smith-Conrad kinetics at low cytochrome c reduction levels, especially in the controlled states. The control of cytochrome oxidase turnover by delta pH and by delta psi can be explained either by an allosteric model or by a model with reversed electron transfer between the binuclear centre and cytochrome a. Other evidence suggests that the reversed electron transfer model may be the correct one.

The proteoliposomal steady state. Effect of size, capacitance and membrane permeability on cytochrome-oxidase-induced ion gradients

1. The flux pathways for H+ and K+ movements into and out of proteoliposomes incorporating cytochrome c oxidase have been investigated as a function of the electrical and geometrical properties of the vesicles. 2. The respiration-induced pH gradient (delta pH) and membrane potential (delta psi) are mutually dependent and individually sensitive to the permeability properties of the membrane. A lowering or abolition of delta psi by the addition of valinomycin increased the steady-state level of delta pH. Conversely, removal of delta pH by the addition of nigericin resulted in a higher steady-state delta psi. 3. Vesicles prepared by sonication followed by centrifugation maintained similar pH gradients at steady state to those in vesicles prepared by dialysis, although the time taken to reach steady state was longer. Higher pH gradients can be induced in non-centrifuged sonicated preparations. 4. No significant differences were found in H+ and K+ permeability between proteoliposomes prepared by dialysis or by sonication. The permeability coefficient of the vesicle bilayers for H+ was 6.1 x 10(-4) cm.s-1 and that for K+ was 7.5 x 10(-10) cm.s-1. An initial fast change in internal pH was seen on the addition of external acid or alkali, followed by a slower, ionophore-sensitive, change. The initial fast phase can be increased by the lipid-soluble base dibucaine and the weak acid oleate. In the absence of ionophores, increasing concentrations of oleate increased the rate of H+ translocation to a level similar to that seen in the presence of nigericin. Internal alkalinization could also be induced by oleate upon the addition of potassium sulphate. 5. The initial, pre-steady-state and steady-state delta pH and delta psi changes can be simulated using a model in which the enzyme responds to both delta pH and delta psi components of the protonmotive force. At steady state, the electrogenic entry of K+ is countered by electroneutral exit via a K+/H+ exchange. 6. The permeability coefficient, PH, calculated from H+ flux under steady-state turnover conditions, was approx. 100 times higher than the corresponding 'passive' measurements of PH. Under conditions of oxidase turnover, the vesicles appear to be intrinsically more permeable to protons.

Use of Oxonol V as a probe of membrane potential in proteoliposomes containing cytochrome oxidase in the submitochondrial orientation

Absorbance changes in the anionic dye bis[3-phenyl-5-oxoisoxazol-4-yl]pentamethineoxonol (oxonol V) can be used to monitor the membrane potential of liposomes and cytochrome c containing cytochrome oxidase proteoliposomes (c-loaded COV). Diffusion potentials (positive inside the vesicles) cause an increase in the dye extinction, with a maximum at 640 nm. A similar increase is seen upon energization of internally facing cytochrome oxidase molecules in c-loaded COV. Both "passive" and "active" responses are only seen when the dye is fully bound to the vesicle membrane. Calibration curves using potassium or n-butyltriphenylphosphonium ion (BTPP+) diffusion potentials are linear up to 100 mV and pass through the origin. Diffusion potentials (positive inside) also cause an increase and red shift in the oxonol V fluorescence emission spectrum. However, potentials of the same sign induced by cytochrome oxidase turnover induce a large fluorescence quenching in c-loaded COV. A similar anomaly has been observed with submitochondrial particles [Smith, J. C., Russ, P., Cooperman, B. S., & Chance, B. (1976) Biochemistry 15, 5094-5105]. A model is proposed consistent with these responses. It is suggested that the dye molecules move further into the membrane phase upon energization, causing the absorbance increase. In the presence of active enzyme, anionic dye molecules are attracted to a positive dipole on each enzyme molecule, causing self-quenching of the fluorescence.

Patterns of O2-dependence of metabolism.

1. In O2 regulating systems, mitochondrial O2 uptake is stabilized as O2 availability declines by means of metabolite signals that simultaneously activate glycolysis; the consequent Pasteur effect is an attempt to make up the energy deficit arising from O2 limitation. 2. In O2 conforming systems, the regulatory link between the ETS and glycolysis is seemingly lost. The advantage of O2 conformity is that it avoids the Pasteur effect; the cost is an exaggerated dependence of mitochondrial respiration on O2 availability. 3. The VO2(max) of man and other low-altitude adapted animals follows the O2 conforming pattern; at altitudes equivalent to the peak of Everest, the VO2(max) is only slightly greater than RMR. Again, key regulatory interactions between the ETS and glycolysis seem to be missing, so the energy deficit is tolerated (lactate production during exercise to exhaustion is less in hypobaric hypoxia than in normoxia). 4. The O2 conformity of VO2(max) in mammals may be explained by inherently inefficient O2 delivery systems in which low Km and low kcat cytochrome oxidase function would be selected. O2-limited maximum mitochondrial respiration helps to explain what would otherwise be a perplexing observation: why over a 10(4) range of mass-specific muscle metabolic rates, the peak O2 uptake rates per unit mitochondrial volume are always the same at VO2(max). 5. The concept of O2-limited mitochondrial respiration predicts that more efficient O2 delivery systems, such as tracheoles found in insect flight muscles, should support much higher in vivo cytochrome oxidase turnover rates. As far as can be currently evaluated, this prediction is realized.(ABSTRACT TRUNCATED AT 250 WORDS)

Extended X-ray absorption fine structure studies of transient species during xanthine oxidase turnover by using rapid freezing

Conference Article| June 01 1986 Extended X-ray absorption fine structure studies of transient species during xanthine oxidase turnover by using rapid freezing G. N. GEORGE; G. N. GEORGE *School of Chemistry and Molecular Sciences, University of Sussex, Brighton BN1 9QJ, Sussex, U.K. Search for other works by this author on: This Site PubMed Google Scholar R. C. BRAY; R. C. BRAY *School of Chemistry and Molecular Sciences, University of Sussex, Brighton BN1 9QJ, Sussex, U.K. Search for other works by this author on: This Site PubMed Google Scholar S. P. CRAMER S. P. CRAMER †Exxon Research and Engineering Company, Annadale, NJ 08801, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1986) 14 (3): 651–652. https://doi.org/10.1042/bst0140651 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation G. N. GEORGE, R. C. BRAY, S. P. CRAMER; Extended X-ray absorption fine structure studies of transient species during xanthine oxidase turnover by using rapid freezing. Biochem Soc Trans 1 June 1986; 14 (3): 651–652. doi: https://doi.org/10.1042/bst0140651 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: EXAFS, extended X-ray absorption fine structure This content is only available as a PDF. © 1986 Biochemical Society1986 Article PDF first page preview Close Modal You do not currently have access to this content.

Peroxide interaction with pulsed cytochrome oxidase. Optical and EPR studies.

EPR and optical analysis of the 420 nm form of cytochrome oxidase (Kumar, C., Naqui, A., and Chance, B. (1984) J. Biol. Chem. 259, 2073-2076) shows that 1) the 420 nm form possesses a 605 nm band, g = 5 EPR signals, and a slightly blue shifted 655 nm band; 2) the reaction of H2O2 with the 420 nm form generates the peroxide complex (Soret band at 427 nm) with the formation of a 580 nm band and abolition of both the 655 nm band and the g = 5 EPR signal. Comparison of our results with past data shows that various forms of oxidase formed from the resting oxidase through different protocols may be identified to be either the 420 nm or the 427 nm form and leads to identification of a peroxy intermediate during oxidase turnover.

Cytochrome o type oxidase from Escherichia coli. Characterization of the enzyme and mechanism of electrochemical proton gradient generation.

Cytochrome o type oxidase purified from the membrane of Escherichia coli consists of four polypeptides (Mr 66000, 35000, 22000, and 17000), and the monomeric form predominates in octyl beta-D-glucopyranoside. The oxidase complex contains two b-type cytochromes (b-558 and b-563) and 2 mol of heme/mol of enzyme. Cytochrome o utilizes ubiquinol-1 and a number of other artificial electron donors as substrates but does not oxidize reduced cytochrome c or ferrocyanide. Activity is highly dependent upon exogenous phospholipids and/or Tween 20, and the quinone analogues 2-heptyl-4-hydroxyquinoline N-oxide and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole are potent inhibitors. Proteoliposomes were formed by detergent dilution or dialysis in the presence of the oxidase and phospholipids, followed by freeze-thaw/sonication. Vesicles formed by this means are unilamellar and contain a random distribution of 85-90-A intramembranous particles on the convex and concave fracture surfaces. During oxidase turnover, the reconstituted system generates a proton electrochemical gradient (interior negative and alkaline) of -115 to -140 mV; however, respiratory control is minimal (i.e., respiratory control ratios of about 1.5 are observed). By using a glass electrode to measure changes in external pH and the fluorescence of entrapped 8-hydroxy-1,3,6-pyrenetrisulfonate to measure changes in internal pH, it is apparent that during ubiquinol oxidation, protons are released on the external surface of the membrane and consumed on the internal surface. In contrast, with N,N,N',-N'-tetramethyl-p-phenylenediamine, an electron donor that carries few protons at neutral pH, little change in external pH is observed until the protonophore carbonyl cyanide m-chlorophenylhydrazone is added, at which point the medium becomes alkaline. The results taken as a whole are consistent with the concept that oxidase turnover generates an electrical potential (interior negative) due to vectorial electron flow from the outer to the inner surface of the membrane. The pH gradient (interior alkaline), on the other hand, appears to result from scalar (i.e., nonvectorial) reactions that consume and release protons at the inner and/or outer surfaces of the membrane, respectively. In other words, cytochrome o oxidase from Escherichia coli does not appear to catalyze vectorial proton translocation.

Reconstitution of b-type cytochrome oxidase from Rhodopseudomonas capsulata in liposomes and turnover studies of proton translocation

Purified b-type cytochrome oxidase from Rhodopseudomonas capsulata was incorporated into phospholipid vesicles to measure proton extrusion with pulses of ferrocytochrome c for one oxidase turnover. In accordance with the pH shift of its midpoint potential, the purified oxidase showed a proton extrusion of 0.24 H + e − with uptake of 1 H + e − from the liposomes for the reduction of oxygen to water. This proton translocation could only be observed in the presence of valinomycin +K + and was not inhibited by DCCD. Oxidase preparations from the first purification step, which contain other protein compounds especially a membrane-bound cytochrome c but not the ubiquinol-cytochrome c 2-oxidoreductase showed a pumping activity of 0.9 H + e − , which was inhibited by DCCD for nearly 75%. Inhibition of the electron transfer was not observed, which could be explained by a ‘molecular slipping’ of proton extrusion and electron transfer. Proton extrusion from two oxidase-turnovers was only 80% of that from one turnover. The proton pumping of the b-type oxidase strongly depended on the enzyme/phospholipid ratio.

A quantitative characterisation of H + translocation by cytochrome c oxidase vesicles

A quantitative analysis of H + extrusion by reconstituted cytochrome c oxidase vesicles is presented with particular regard to the decay kinetics of the extruded proton pulse and to the structural heterogeneity of the vesicle preparation. The decay of the extruded H + pulse under conditions typical of those used for its measurement is much slower than expected from the passive proton permeability of the vesicle membranes. It is shown that this apparent anomaly results from insufficient transmembrane charge equilibration via valinomycin and K + during oxidase turnover. This situation can be remedied by increasing the valinomycin concentration or by replacing this counterion system with 1 mM tetraphenylphosphonium. Under these latter conditions, the decay kinetics can be described as the sum of two exponential terms. To facilitate interpretation of the proton pump decay kinetics, a structural analysis of the oxidase vesicle preparation is presented. The bulk of the reconstituted vesicles (i.e., those representing approx. 80% of the total oxidase and lipid) are 30–62 nm in diameter. At least 70% of the reconstituted oxidase molecules are contained individually in separate vesicles, indicating that the enzyme monomer is competent in H + translocation.