Low-temperature flash photolysis studies of cytochrome oxidase and its environment

The CO-binding kinetics of cytochrome a₃ in isolated, detergent-solubilized cytochrome oxidase have been studied by flash photolysis over wide ranges of CO concentration and temperature. The results strongly suggest that CO has an intermediate bound state in its path to the final bound state at the heme iron. In the temperature range 230–273 K in frozen aqueous solutions, the recombination rates depend upon CO concentration; at low CO concentrations the kinetics are biphasic. The rate of the faster process depends upon the detergent concentration, that of the slower process upon the salt concentration. In addition, the faster process depends upon the amount of CO photodissociated. It is concluded that the cytochrome oxidase molecules are aggregated in regions that contain detergent and possibly some lipids. These regions retain considerable fluid character well below the macroscopic freezing point of the solution. The faster phase of the recombination is interpreted as the rebinding of CO molecules that remain in the fluid region after photodissociation. The slower phase would then be due to the migration of some dissociated CO out into the surrounding frozen solvent. The non-Arrhenius behavior of both phases probably represents partial melting of the medium; preliminary NMR measurements of mobile protons support this hypothesis. Many of the kinetic features described here are also seen in mitochondria; thus the detergent-solubilized cytochrome oxidase may be a useful model system for the ligand-binding behavior of the enzyme in the mitochondrial membrane.     

The reaction of Pseudomonas aeruginosa cytochrome c oxidase with carbon monoxide.

The binding of CO to ascorbate-reduced Pseudomonas cytochrome oxidase was investigated by static-titration, stopped-flow and flash-photolytic techniques. Static-titration data indicated that the binding process was non-stoicheiometric, with a Hill number of 1.44. Stopped-flow kinetics obtained on the binding of CO to reduced Pseudomonas cytochrome oxidase were biphasic in form; the faster rate exhibited a linear dependence on CO concentration with a second-order rate constant of 2 X 10(4) M-1-s-1, whereas the slower reaction rapidly reached a pseudo-first-order rate limit at approx. 1s-1. The relative proportions of the two phases observed in stopped-flow experiments also showed a dependency on CO concentration, the slower phase increasing as the CO concentration decreased. The kinetics of CO recombination after flash-photolytic dissociation of the reduced Pseudomonas cytochrome oxidase-CO complex were also biphasic in character, both phases showing a linear pseudo-first-order rate dependence on CO concentration. The second-order rate constants were determined as 3.6 X 10(4)M-1-s-1 and 1.6 X 10(4)M-1-s-1 respectively. Again the relative proportions of the two phases varied with CO concentration, the slower phase predominating at low CO concentrations. CO dissociation from the enzyme-CO complex measured in the presence of O2 and NO indicated the presence of two rates, of the order of 0.03s-1 and 0.15s-1. When sodium dithionite was used as a reducing agent for the Pseudomonas cytochrome oxidase, the CO-combination kinetics observed by both stopped flow and flash photolysis were extremely complex and not able to be simply analysed.     

Identification of 27-oxo-octacosanoic acid and heptacosane-1,27-dioic acid in Legionella pneumophila

A strain of Escherichia coli having elevated levels of cytochrome bo and lacking the cytochrome bd quinol oxidase was grown in chemostat culture at low copper levels. Such cells had lowered levels of copper and of total cytochrome b. Cytochrome o concentration was unchanged when assayed by conventional CO difference spectroscopy, but apparently diminished by 80% in copper-deficient cells as determined by photodissociation of bound CO at 193 K. This is attributed to depletion of copper in the oxidase of copper-deficient cells, causing rapid recombination of photodissociated CO to haem O. CO recombination was also more sensitive to low intensities of actinic light in copper-depleted oxidase. The results illustrate a further similarity between the active sites of o- and aa3-type terminal oxidases.     

The effect of detergents on bovine cytochrome c oxidase: a kinetic approach

(1) Investigation of the relationship between the detergent concentration and steady-state and pre-steady-state kinetics of cytochrome c oxidase proved to be a valid approach in the study of protein-detergent interaction. (2) Laurylmaltoside, sodium cholate and Triton X-100 influenced the kinetics of cytochrome c oxidase cooperatively at detergent concentrations near their critical micelle concentration. This mode of interaction reflects disaggregation of the oxidase as a result of cooperative binding of the detergent. (3) Addition of increasing concentrations of Tween-80 to the aggregated enzyme caused a more gradual decrease in aggregation of the oxidase, which did not result in a change in activity of the enzyme. This suggests that aggregation of cytochrome c oxidase occurs in a highly regular manner in which no catalytic sites are shielded off. (4) Oxidase aggregates present at detergent concentrations below the critical micelle concentration of laurylmaltoside and Triton X-100 showed considerable activity. Their kinetics were equal to those of the oxidase in Tween-80, suggesting that the protein molecules are aligned in a similar way in all oligomers. Aggregates present in low concentrations of sodium cholate showed turnover rates that were twice as low as those observed with other aggregates. (5) Solubilisation of the oxidase by sodium cholate or Triton X-100 resulted in almost complete inhibition of enzymic activity, whereas the association rate of ferrocytochrome c was almost equal to that found for monomeric oxidase in laurylmaltoside. These results are in agreement with a mixed-type inhibition.     

Binding of homogeneous cytochrome b5 to rat liver microsomes. Effect on N-demethylation reactions.

Incubation of rat homogeneous detergent-solubilized cytochrome b5 with rat liver microsomes resulted in specific binding of the hemoprotein which was rapidly reduced by NADH. The NADH cytochrome c reductase activity in these preparations increased in proportion to the amount of cytochrome bound. However, the extra-bound detergent-solubilized cytochrome b5 did inhibit NADPH-dependent N-demethylations, the NADH synergism and NADPH cytochrome P-450 reductase activity. Manganese protoporphyrin-apocytochrome complex when bound to microsomes in amounts equivalent to detergent-solubilised cytochrome b5 showed no effect on N-demethylation activity. Furthermore, the binding of cytochrome b5 preparations reconstituted from heme and apocytochrome b5 had no effect on either the NADPH-dependent N-demethylation of aminopyrene or ethylmorphine or the NADH synergism observed with rat liver microsomes. In addition, homogeneous cytochrome b5 eluted from three additional Sephadex G-100 columns showed no inhibitory effects when bound to liver microsomes. Spectral analyses of the acid-acetone extract of the hemoprotein showed an absorption peak at 278 nm suggesting that the homogeneous b5 contains contaminating amounts of tightly bound detergent which is responsible for the observed inhibition of mixed function oxidase activity and which is removed during extraction of the heme from the apocytochrome and during further gel filtration applications.     

Cytochrome c and c2 binding dynamics and electron transfer with photosynthetic reaction center protein and other integral membrane redox proteins

To further the understanding of the details of c-type cytochrome action as a redox carrier between major electron-transfer proteins, the single-turnover kinetics time course of cytochrome c and cytochrome c2 oxidation by flash-activated photosynthetic reaction center (purified from the bacterium Rhodobacter sphaeroides) has been examined under a wide variety of conditions of concentration, ionic strength, and viscosity with reaction center present in detergent dispersion and phosphatidylcholine proteoliposomes. We find that the three-state model proposed by Overfield and Wraight [Overfield, R. E., & Wraight, C. A. (1980) Biochemistry 19, 3322-3327] is generally sufficient to model the kinetics time course; many similarities are found with the cytochrome c-cytochrome c oxidase interaction in mitochondria. Further, we find the following: (1) Significant "product inhibition" by oxidized cytochrome c (c2) bound to the reaction center is apparent. (2) The viscosity sensitivity of the electron transfer into the reaction center from bound cytochrome c (c2) suggests a physical interpretation of the distal state. (3) The exchange dynamics of oxidized and reduced cytochrome c (c2) are similar regardless of the state of activation of the reaction center. (4) Preferential binding of the oxidized form of cytochrome c is revealed upon reconstitution of the reaction center into neutral lipid vesicles, permitting an independent confirmation of the binding suggested by the kinetics. (5) Flash-activated electron-transfer kinetics in reaction center hybrid protein systems have shown that diffusion and competitive binding characterize the behavior of cytochrome c as a redox carrier between the reaction center protein and either the cytochrome bc1 complex or the cytochrome c oxidase.     

Kinetics of the reactions of trioxodinitrate and nitrite ions with cytochrome d in Escherichia coli.

The rate of reaction of trioxodinitrate with reduced cytochrome oxidase d in membrane particles from Escherichia coli at pH 7 and 25 degrees C depends linearly upon [HN2O3-] over the concentration range studied (up to 0.05 mM) and is also first-order in cytochrome d. The known rate of decomposition of trioxodinitrate to give NO- and NO2- is about 4.5-times faster than the rate of reaction of reduced cytochrome d with trioxodinitrate, implying that cytochrome d reacts directly with NO-, with a trapping ratio of between 0.20 and 0.25, rather than with trioxodinitrate. The implications of the facile formation of the NO(-)-nitrosyl complex of cytochrome d for the mechanism of denitrification are discussed with particular reference to the mechanism of N-N bond formation. The reaction of reduced cytochrome d with nitrite (a decomposition product of trioxodinitrate) under these conditions is much slower than that with trioxodinitrate. The kinetics show a biphasic dependence of initial rate upon nitrite concentration. The rate data at low [NO2-] are consistent with saturation of a high affinity site for nitrite, having Vmax = 4.29.10(-9) M s-1 and Km = 0.034 mM. The existence of two binding sites for nitrite is consistent with the suggestion that the cytochrome bd complex contains two cytochrome d haems.     

Reaction of oxidized cytochrome oxidase with cyanide. Effects of pH, cytochrome c and membrane environment

Binding of HCN with ferric beef heart cytochrome oxidase has been studied in submitochondrial particles, as with the enzyme solubilized in detergent or reconstituted into proteoliposomes. Under all conditions, the reaction proceeds via an intermediate and its kinetics can be described by formal parameters Km and kmax in keeping with the Michaelis-type equation. Km of the reaction strongly depends on the enzyme environment; thus it increases 100-1000 fold upon solubilization of cytochrome oxidase but can be subsequently decreased by incorporation of the enzyme in liposomes and by addition of cytochrome c. pH-dependence of the reaction rate shows that, in submitochondrial particles and proteoliposomes as well as in the case of solubilized enzyme supplement with cytochrome c, HCN specifically binds the form of cytochrome oxidase in which a heme-linked ionizable group with pKa 6,5-6,9 is protonated.     

Triton X-100 induced dissociation of beef heart cytochrome c oxidase into monomers

Purified beef heart cytochrome c oxidase, when solubilized with at least 5 mg of Triton X-100/mg of protein, was found to be a monodisperse complex containing 180 molecules of bound Triton X-100 with a protein molecular weight of 200 000, a Stokes radius of 66-72 A, and an s(0)20,w = 8.70 S. These values were determined by measurement of the protein molecular weight by sedimentation equilibrium in the presence of D2O, evaluation of the sedimentation coefficient, S(0)20,w, by sedimentation velocity with correction for its dependence upon the concentration of protein and detergent, and measurement of the effective radius by calibrated Sephacryl S-300 gel chromatography. The monomeric complex was judged to be homogeneous and monodisperse since the effective mass of the complex was independent of the protein concentration throughout the sedimentation equilibrium cell and a single protein schlieren peak was observed during sedimentation velocity. These results are interpreted in terms of a fully active monomeric complex that exhibits typical biphasic cytochrome c kinetics and contains 2 heme a groups and stoichiometric amounts of the 12 subunits normally associated with cytochrome c oxidase. With lower concentrations of Triton X-100, cytochrome c oxidase dimers and higher aggregates can be present together with the monomeric complex. Monomers and dimers can be separated by sedimentation velocity but cannot be separated by Sephacryl S-300 gel filtration, probably because the size of the Triton X-100 solubilized dimer is not more than 20% larger than the Triton X-100 solubilized monomer.(ABSTRACT TRUNCATED AT 250 WORDS)     

The aggregation state of bovine heart cytochrome c oxidase and its kinetics in monomeric and dimeric form

The monomeric and dimeric forms of bovine cytochrome c oxidase (EC 1.9.3.1) were obtained from gel filtration chromatography on Ultrogel AcA 34 and analyzed. Both species contained all 12-13 subunits described for this enzyme. In the dimer 320 molecules [3H]dodecyl-beta-D-maltoside were bound per heme aa3 and in the monomer 360 molecules per heme aa3. The monomers contained 10 mol of tightly bound phospholipid/mol heme aa3 and the dimers 14. Sedimentation coefficients of 15.5-18 S for the dimer and 9.6 S for the monomer were calculated from sucrose density centrifugation analysis and analytical centrifugation. By the laser beam light-scattering technique a Stokes radius of 70 A for the dimeric detergent-lipid-protein complex was measured. From those parameters and the densitometric determined partial specific volumes of the detergent and the enzyme, the molecular weights of 400,000 for the protein moiety of the dimer and 170,000-200,000 for the monomer were calculated. Under very low ionic strength conditions the monomer/dimer equilibrium was found to be dependent on the protein concentration. At low enzyme concentrations (10(-9) M) monomers were predominant, whereas at concentrations above 5 X 10(-6) M the amounts of dimers and higher aggregates were more represented. The cytochrome c oxidase activity, measured spectrophotometrically and analyzed by Eadie-Hofstee plot, was biphasic as a function of cytochrome c concentration for the dimeric enzyme. Pure monomers gave monophasic kinetics. The data, fitting with a homotropic negative cooperative mechanism for the dimer of cytochrome c oxidase, are discussed and compared with other described mechanisms.     

Chromous ion reduction of mammalian cytochrome oxidase and some of its derivatives.

The reduction of cytochrome c oxidase by Cr2+, followed by means of stopped-flow spectrophotometry, exhibits two phases: the faster Cr2+-concentration-dependent reaction has an initial rate constant of 1.1 X 10(4)M-1-S-1, but reaches a rate limit at high concentration of reductant; the slower phase is concentration-independent with a rate of 0.3S-1. The activation energies of the fast and the slow processes are 35 and 71 kJ/mol respectively. The reduction kinetics of the mixed-valence CO complex and the cyanide-inhibited enzyme were compared with those of the fully oxidized forms: both the liganded species have a fast phase identical with that found in the oxidized oxidase. A comparison of the kinetic difference spectra obtained for the fast phase of reduction of oxidized oxidase with those obtained on reduction of the liganded species suggests that the rapid phase arises from the reduction ofhaem a, and the slow phase from the reduction of haem a3.     

The effect of mitochondrial energization on cytochrome c oxidase kinetics as measured at low temperatures. I. The reaction with carbon monoxide.

The kinetics of CO binding by the cytochrome c oxidase of pigeon heart mitochondria were studied as a function of membrane energization at temperatures from 180 to 280 degrees K in an ethylene glycol/water medium. Samples energized by ATP showed acceleration of CO binding compared to those untreated or uncoupled by carbonylcyanide p-trifluoromethyoxyphenylhydrazone but only at relatively low temperatures and high CO concentrations. Experiments using samples in a "mixed valency" (partially oxidized) state showed that the acceleration of ligand binding is not due to the formation of a partially oxidized state via reverse electron transport. It is concluded that in the deenergized state one CO molecule can be closely associated with the cytochrome a3 heme site without actually being bound to the heme iron; in the energized state, two or more ligand molecules can occupy this intermediate position. The change in the apparent ligand capacity of a region near the heme iron in response to energization is evidence for an interaction between cytochrome oxidase and the ATPase system. Furthermore, these results suggest a control mechanism for O2 binding.     

ATP induces conformational changes in mitochondrial cytochrome c oxidase. Effect on the cytochrome c binding site

ATP influences the kinetics of electron transfer from cytochrome c to mitochondrial oxidase both in the membrane-embedded and detergent-solubilized forms of the enzyme. The most relevant effect is on the so-called "high affinity" binding site for cytochrome c which can be converted to "low affinity" by millimolar concentrations of ATP (Ferguson-Miller, S., Brautigan, D. L., and Margoliash, E. (1976) J. Biol. Chem. 251, 1104-1115). This phenomenon is characterized at the molecular level by the following features. ATP triggers a conformational change on the water-exposed surface of cytochrome c oxidase; in this process, carboxyl groups forming the cluster of negative charges responsible for binding cytochrome c change their accessibility to water-soluble protein modifier reagents; as a consequence the electrostatic field that controls the enzyme-substrate interaction is altered and cytochrome c appears to bind differently to oxidase; photolabeling experiments with the enzyme from bovine heart and other eukaryotic sources show that ATP cross-links specifically to the cytoplasmic subunits IV and VIII. Taken together, these data indicate that ATP can, at physiological concentration, bind to cytochrome c oxidase and induce an allosteric conformational change, thus affecting the interaction of the enzyme with cytochrome c. These findings raise the possibility that the oxidase activity may be influenced by the cell environment via cytoplasmic subunit-mediated interactions.     

Control of electron transfer by the electrochemical potential gradient in cytochrome-c oxidase reconstituted into phospholipid vesicles

The kinetics of electron transfer between cytochrome-c oxidase and ruthenium hexamine has been characterized using the native enzyme or its cyanide complex either solubilized by detergent (soluble cytochrome oxidase) or reconstituted into artificial phospholipid vesicles (cytochrome oxidase-containing vesicles). Ru(NH3)2+6 (Ru(II] reduces oxidized cytochrome a, following (by-and-large) bimolecular kinetics; the second order rate constant using the cyanide complex of the enzyme is 1.5 x 10(6) M-1 s-1, for the enzyme in detergent, and slightly higher for COV. In the case of COV the kinetics are not affected by the addition of ionophores. Upon mixing fully reduced cytochrome oxidase with oxygen (in the presence of excess reductants), the oxidation leading to the pulsed enzyme is followed by a steady state phase and (eventually) by complete re-reduction. When the concentrations of dioxygen and oxidase are sufficiently low (micromolar range), the time course of oxidation can be resolved by stopped flow at room temperature, yielding an apparent bimolecular rate constant of 5 x 10(7) M-1 s-1. After exhaustion of oxygen and end of steady state, re-reduction of the pulsed enzyme by the excess Ru(II) is observed; the concentration dependence shows that the rate of re-reduction is limited at 3 s-1 in detergent; this limiting value is assigned to the intramolecular electron transfer process from cytochrome a-Cua to the binuclear center. Using the reconstituted enzyme, the internal electron transfer step is sensitive to ionophores, increasing from 2-3 to 7-8 s-1 upon addition of valinomycin and carbonyl cyanide m-chlorophenylhydrazone. This finding indicates for the first time an effect of the electrochemical potential across the membrane on the internal electron transfer rate; the results are compared with expectations based on the hypothesis formulated by Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), and their bioenergetic relevance is discussed with reference to the proton pumping activity of the enzyme.     

Kinetic investigations of the reactions of cytochrome c oxidase with hydrogen peroxide

The reaction of H2O2 with reduced cytochrome c oxidase was investigated with rapid-scan/stopped-flow techniques. The results show that the oxidation rate of cytochrome a3 was dependent upon the peroxide concentration (k = 2 X 10(4) M-1 X s-1). Cytochrome a and CuA were oxidised with a maximal rate of approx. 20 s-1, indicating that the rate of internal electron transfer was much slower with H2O2 as the electron acceptor than with O2 (k greater than or equal to 700 s-1). Although other explanations are possible, this result strongly suggests that in the catalytic cycle with oxygen as a substrate the internal electron-transfer rate is enhanced by the formation of a peroxo-intermediate at the cytochrome a3-CuB site. It is shown that H2O2 took up two electrons per molecule. The reaction of H2O2 with oxidised cytochrome c oxidase was also studied. It is shown that pulsed oxidase readily reacted with H2O2 (k approximately 700 M-1 X s-1). Peroxide binding is followed by an H2O2-independent conformational change (k = 0.9 s-1). Resting oxidase partially bound H2O2 with a rate similar to that of pulsed oxidase; after H2O2 binding the resting enzyme was converted into the pulsed conformation in a peroxide-independent step (k = 0.2 s-1). Within 5 min, 55% of the resting enzyme reacted in a slower process. We conclude from the results that oxygenated cytochrome c oxidase probably is an enzyme-peroxide complex.     

Phospholipid composition and organization of cytochrome c oxidase preparations as determined by 31P-nuclear magnetic resonance

The molecular organization as well as the composition of the phospholipids in cytochrome c oxidase preparations (bovine heart) were investigated by 31P-nuclear magnetic resonance. In the so-called 'lipid-rich' preparation the lipids were found to form a fluid bilayer around the enzyme since the 31P-NMR spectrum was characteristic of a fast, axially symmetric motion of the phosphate groups with a chemical shift anisotropy of delta sigma = -45 ppm. In contrast, the 'lipid-depleted' cytochrome c oxidase gave rise to a broader spectrum where the motion of the phospholipids was no longer axially symmetric. Nevertheless, the total width of the spectrum was still considerably narrower than observed for immobilized phospholipids in solid crystals. Both enzyme preparations were dissolved in 1% detergent solution and used for high-resolution 31P-NMR spectroscopy. Narrow lines of about 20 Hz linewidth were obtained for both types of enzyme preparations, and well-resolved resonances could be assigned to cardiolipin, phosphatidylethanolamin and phosphatidylcholine. The major differences between lipid-rich and lipid-depleted cytochrome c oxidase were the absolute amount of phospholipid associated with the protein and the relative contribution of the individual lipid classes to the 31P-NMR spectrum. For lipid-rich cytochrome c oxidase about 130 molecules phospholipid were bound per enzyme (approx. 11 cardiolipins, 54 phosphatidylethanolamines and 64 phosphatidylcholines). For lipid-depleted cytochrome c oxidase only 6-18 lipids were bound per enzyme (1 or 2 cardiolipins, 3-8 phosphatidylethanolamines and 2-8 phosphatidylcholines). In contrast to earlier suggestions that cardiolipin is the only remaining lipid in lipid-depleted cytochrome c oxidase, the 31P-NMR studies demonstrate that all three lipids remain associated with the protein.     

The interaction of quinone and detergent with reaction centers of purple bacteria. I. Slow quinone exchange between reaction center micelles and pure detergent micelles.

The kinetics of light-induced electron transfer in reaction centers (RCs) from the purple photosynthetic bacterium Rhodobacter sphaeroides were studied in the presence of the detergent lauryldimethylamine-N-oxide (LDAO). After the light-induced electron transfer from the primary donor (P) to the acceptor quinone complex, the dark re-reduction of P+ reflects recombination from the reduced acceptor quinones, QA- or QB-. The secondary quinone, QB, which is loosely bound to the RC, determines the rate of this process. Electron transfer to QB slows down the return of the electron to P+, giving rise to a slow phase of the recovery kinetics with time tau P approximately 1 s, whereas charge recombination in RCs lacking QB generates a fast phase with time tau AP approximately 0.1 s. The amount of quinone bound to RC micelles can be reduced by increasing the detergent concentration. The characteristic time of the slow component of P+ dark relaxation, observed at low quinone content per RC micelle (at high detergent concentration), is about 1.2-1.5 s, in sharp contrast to expectations from previous models, according to which the time of the slow component should approach the time of the fast component (about 0.1 s) when the quinone concentration approaches zero. To account for this large discrepancy, a new quantitative approach has been developed to analyze the kinetics of electron transfer in isolated RCs with the following key features: 1) The exchange of quinone between different micelles (RC and detergent micelles) occurs more slowly than electron transfer from QB- to P+; 2) The exchange of quinone between the detergent "phase" and the QB binding site within the same RC micelle is much faster than electron transfer between QA- and P+; 3) The time of the slow component of P+ dark relaxation is determined by (n) > or = 1, the average number of quinones in RC micelles, calculated only for those RC micelles that have at least one quinone per RC (in excess of QA). An analytical function is derived that relates the time of the slow component of P+ relaxation, tau P, and the relative amplitude of the slow phase. This provides a useful means of determining the true equilibrium constant of electron transfer between QA and QB (LAB), and the association equilibrium constant of quinone binding at the QB site (KQ+). We found that LAB = 22 +/- 3 and KQ = 0.6 +/- 0.2 at pH 7.5. The analysis shows that saturation of the QB binding site in detergent-solubilized RCs is difficult to achieve with hydrophobic quinones. This has important implications for the interpretation of apparent dependencies of QB function on environmental parameters (e.g. pH) and on mutational alterations. The model accounts for the effects of detergent and quinone concentration on electron transfer in the acceptor quinone complex, and the conclusions are of general significance for the study of quinone-binding membrane proteins in detergent solutions.     

A new approach for studying fast biological reactions involving dioxygen: the reaction of fully reduced cytochrome c oxidase with O2

We describe a new method for studying rapid biological reactions involving dioxygen. This approach is based on the photolysis of a synthetic caged dioxygen carrier, which produces dioxygen on a fast time scale. The method was used to investigate the reduction of dioxygen to water by cytochrome c oxidase at room temperature following photolysis of a (mu-peroxo)(mu-hydroxo)bis[bis(bipyridyl)c obalt(III)] complex. The fact that dioxygen is generated in situ on a nanosecond or faster time scale avoids potential complications related to the fate of photodissociated CO in a conventional CO flow-flash experiment. The cobalt complex is stable at room temperature under anaerobic conditions and releases dioxygen upon irradiation at 355 nm with a quantum yield of 0.04. The complex does not react with reduced cytochrome oxidase or its reducing agents within the mixing time of the experiment, and its photoproducts do not interfere with the kinetics of the dioxygen reduction. The oxidation of the reduced cytochrome oxidase was monitored between 500 and 750 nm using a gated optical spectrometric multichannel analyzer following photodissociation of the cobalt complex. The data were analyzed using singular value decomposition and global exponential fitting, and two apparent lifetimes (380 +/- 50 micros and 1.7 +/- 0.2 ms) were resolved and compared to results from a conventional CO flow-flash experiment. The results show that approximately 90 microM dioxygen can be generated upon a single laser pulse and that this approach can be used to study other fast biological reactions involving O(2).     

Electron redistribution in mixed valence cytochrome oxidase following photolysis of carboxy-oxidase

Absorbance changes at 446 nm in purified cytochrome oxidase following flash photolysis of carboxy-oxidase poised in the mixed valence state at +220 mV show biphasic kinetics. One phase corresponds to CO recombination to ferrous cytochromea ₃ with an energy of activation of 9 kcal/mol; the second phase is 3–5 times faster with an energy of activation of 9.15 kcal/mol. Following flash photolysis at approximately –60°C, cytochromesa andc and the 840-nm CuA species are observed to undergo reduction as electrons from ferrous unliganded cytochromea ₃ equilibrate with the equipotential redox centers of the oxidase; as CO recombines with ferrous cyochromea ₃, these centers are oxidized and the mixed valence carboxy-oxidase is regenerated. Electron redistribution between centers of the oxidase in the forward and reverse directions occurs faster than does the binding of CO.     

Leukotriene C synthase in mouse mastocytoma cells. An enzyme distinct from cytosolic and microsomal glutathione transferases.

The reactions between cellobiose and cellobiose oxidase were investigated by stopped-flow spectrophotometry. Under anaerobic conditions rapid reduction of the associated flavin is followed by slower reduction of cytochrome b. The kinetic difference spectra are reported. The rate of flavin reduction depends on the cellobiose concentration (with an apparent second-order rate constant of approx. 10(5) M-1.s-1) but reaches a rate limit of approx. 20 s-1. In contrast, the rate of cytochrome b reduction decreases at high cellobiose concentrations. Kinetic titrations of the flavin and cytochrome b moieties yield the stoichiometries of the separate reactions, i.e. the number of moles of cellobiose needed to fully reduce 1 mole of each redox component. The rate constant for cytochrome b reduction, unlike that for flavin reduction, increased with enzyme concentration, prompting the conclusion that any given cytochrome b centre is reduced preferentially by flavin groups in different molecules rather than by its partner flavin within the same monomer. These data are discussed in the context of a scheme that rationalizes them and accounts for the overall stoichiometry in which three two-electron donors (cellobiose molecules) reduce two three-electron acceptors (the flavin-cytochrome b of cellobiose oxidase).