Kinetic studies on (N-formyltryptophyl)cytochrome c.

The formylation of the ring nitrogen atom of the tryptophan residue in cytochrome c was carried out and consequent changes in the kinetic properties of the protein were investigated. The reduction of formylated cytochrome c by Cr2+ was studied by stopped-flow techniques. At pH 6.5 the reduction process shows the presence of two phases. One phase (k = 4 X 10(4) M-1-s-1) is dependent on Cr2+ concentration and one phase (k = 5.0 s-1) is not. A study of the temperature dependence of the two phases yields values for their activation energies of 38.6kJ-mol-1 and 42.4kJ-mol-1 respectively. The reaction of the reduced formylated cytochrome c with CO was followed by means of both stopped-flow techniques and flash photolysis. The combination with CO at pH 6.8 measured in stopped-flow experiments shows two phases, both dependent on the concentration of CO (k1 = 1.8 X 10(2) M-1-s-1). If CO was dissociated from the protein by photolysis and then allowed to recombine with it, it was found to do so in a simple manner, at a rate which depended on the concentration of CO (k = 1.9 X 10(2) M-1-s-1). A tentative model which can accommodate these findings is proposed. The reaction of the oxidized form of formylated cytochrome c with NO was followed by means of stopped-flow techniques. The reaction was found to be biphasic with one phase dependent on the concentration of NO (k = 2.8 X 10(3) M-1-s-1) and one phase (k = 0.2x-1) independent of the concentration of NO. This behaviour is compared with that of the native molecule. A comparison of these kinetic observations with those on other tryptophan-specific modifications leads to the conclusion that the main alteration in kinetic properties is due, not to the nature of the modifying group, but rather to the disruption of the normal environment of the haem.     

Kinetic studies on mammalian cytochrome c modified with 2-hydroxy-5-hydroxy-5-nitrobenzyl bromide.

The reduction of 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c by the chromous ion was studied by stopped-flow techniques. At pH6.5 the reduction of 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c is complex, showing the presence of three distinct phases. Two chromium concentration-dependent phases are observed (1.1 X 10(5) M-1-S-1, phase 1; 1.25 X 10(4)M-1-S-1, phase 2) and one slow first-order process (0.25S-1, phase 3). A comparison of the static and kinetic difference spectra, along with the data from the reduction of the reoxidized reduced protein, suggests that the slow chromium concentration-independent phase is due to a slow conformational event after fast reduction of the NO2 group. The rates of the chromium concentration-dependent phases show a marked variation with pH above 7.5. The activation energies for the three processes were also measured at 33.2, 38.6 and 69.7 kJ-mol-1 for phases 1, 2 and 3 respectively. The reaction of reduced 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c with CO was foollowed by means of both stopped-flow and flash photolysis. The combination with CO at pH 6.8 as measured in stopped-flow experiments showed two phases, one CO-dependent phase (phase 2, 2.4 X 10(2)M-1-S-1) and one CO-independent phase (phase 1, 0.015S-1). Investigation of the pH-dependence of the phases showed both the rates and amounts of each phase to be pH-invariant. CO recombination, after photolytic removal, was found to be biphasic; a CO-dependent phase (phase 2, 2.4 X 10(2)M-1-S-1) and a CO-independent phase (phase 1, 1.0s-1) were observed. A tentative model which can accommodate these observations is proposed.     

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.     

The reduction of Pseudomonas cytochrome c551 oxidase by chromous ions.

The reduction of cytochrome c551 oxidase from Pseudomonas aeruginosa by Cr2+ ions was followed in the stopped-flow apparatus at a number of wavelengths. The c-haem reduction proceeded in a biphasic fashion with second-order rate constants of 2.6 X 10(5)M-1-S-1 and 4.8 X 10(4)M-1-S-1 at 25 degrees C, whereas the biphasic reduction of the d1-haem appeared to be independent of reductant concentration with rate constants of approx. 1.0S-1 and 0.25S-1 respectively. The kinetically determined difference spectra (reduced minus oxidized) for the c- and d1-haems are presented.     

Kinetic studies on the binding of cyanide to oxygenated cytochrome c oxidase.

The reaction of cyanide with oxygenated cytochrome c oxidase was followed by means of flow-flash techniques. The oxygenated form, produced after photolysis of the partially reduced CO complex in the presence of cyanide and O2, shows cyanide-binding properties distinct from those of both the oxidized and the reduced forms of the protein. The binding is a single process (k = 22M-1-S-1) linearly dependent on cyanide concentration to as high as 75 mM. It is suggested that the oxygenated form is a conformational variant of the oxidized protein.     

The electron-transfer reaction between azurin and the cytochrome c oxidase from Pseudomonas aeruginosa.

A stopped-flow investigation of the electron-transfer reaction between oxidized azurin and reduced Pseudomonas aeruginosa cytochrome c-551 oxidase and between reduced azurin and oxidized Ps. aeruginosa cytochrome c-551 oxidase was performed. Electrons leave and enter the oxidase molecule via its haem c component, with the oxidation and reduction of the haem d1 occurring by internal electron transfer. The reaction mechanism in both directions is complex. In the direction of oxidase oxidation, two phases assigned on the basis of difference spectra to haem c proceed with rate constants of 3.2 X 10(5)M-1-S-1 and 2.0 X 10(4)M-1-S-1, whereas the haem d1 oxidation occurs at 0.35 +/- 0.1S-1. Addition of CO to the reduced enzyme profoundly modifies the rate of haem c oxidation, with the faster process tending towards a rate limit of 200S-1. Reduction of the oxidase was similarly complex, with a fast haem c phase tending to a rate limit of 120S-1, and a slower phase with a second-order rate of 1.5 X 10(4)M-1-S-1; the internal transfer rate in this direction was o.25 +/- 0.1S-1. These results have been applied to a kinetic model originally developed from temperature-jump studies.     

Nitrogenase of Azotobacter chroococcum. Kinetics of the reduction of oxidized iron-protein by sodium dithionite.

The kinetics of the reduction of oxidized Fe-protein of nitrogenase from Azotobacter chroococcum by sodium dithionite were studied by stopped-flow and rapid-freezing e.p.r. (electron-paramagnetic-resonance) spectroscopy. The appearance of the gav. = 1.94 e.p.r. signal (0.24 electron integrated intensity/mol) was associated with a one-electron reduction by SO2--with k greater than 10(8)M-1-S-1 at 23 degrees C. A value of k = 1.75s-1 was obtained for the rate of dissociation of S2O42- into 2SO2-- at 23 degrees C. Further reductions by SO2-- occurred in three slower phases with rate constants in the range 10(4) -10(6)M-1-S-1. These latter phases have no corresponding e.p.r. signal changes and are probably associated with enzymically inactive protein. The high rate of reduction by SO2-- of the Fe-protein alone (k greater than 10(8)M-1-S-1) relative to the rate of oxidation of the Fe-protein in the catalytically active Fe:Mo-Fe protein complex (k = 2.2 X 1O(2)s-1) and the observation that in the steady state the Fe-protein is substantially oxidized means that at normal assay concentrations another reaction must limit the rate of reduction of Fe-protein during turnover.     

Kinetics of Pseudomonas aeruginosa cytochrome c551 and cytochrome oxidase oxidation by Co(phen)3(3+) and Mn(CyDTA)(H2O)-

The reaction between reduced Pseudomonas cytochrome c551 and cytochrome oxidase with two inorganic metal complexes, Co(phen)3(3+) and Mn(CyDTA)(H2O)-, has been followed by stopped-flow spectrophotometry. The electron transfer to cytochrome c551 by both reactants is a simple process, characterized by the following second-order rate constant: k = 4.8 X 10(4) M-1 sec-1 in the case of Co(phen)3(3+) and k = 2.3 X 10(4) M-1 sec-1 in the case of Mn(CyDTA)(H2O)-. The reaction of the c-heme of the oxidase with both metal complexes is somewhat heterogeneous, the overall process being characterized by the following second-order rate constants: k = 1.7 X 10(3) M-1 sec-1 with Co(phen)3(3+) and k = 4.3 X 10(4) M-1 sec-1 with Mn(CyDTA)(H2O)- as oxidants; under CO (which binds to the d1-heme) the former constant increases by a factor of 2, while the latter does not change significantly. The oxidation of the d1-heme of the oxidase by Co(phen)3(3+) occurs via intramolecular electron transfer to the c-heme, a direct bimolecular transfer from the complex being operative only at high metal complex concentrations; when Mn(CyDTA)(H2O)- is the oxidant, the bimolecular oxidation of the d1-heme competes successfully with the intramolecular electron transfer.     

Nitrogenase of Klebsiella pneumoniae. Kinetic studies on the Fe protein involving reduction by sodium dithionite, the binding of MgADP and a conformation change that alters the reactivity of the 4Fe-4S centre.

The kinetics of reduction of indigocarmine-dye-oxidized Fe protein of nitrogenase from Klebsiella pneumoniae (Kp2ox) by sodium dithionite in the presence and absence of MgADP were studied by stopped-flow spectrophotometry at 23 degrees C and at pH 7.4. Highly co-operative binding of 2MgADP (composite K greater than 4 X 10(10) M-2) to Kp2ox induced a rapid conformation change which caused the redox-active 4Fe-4S centre to be reduced by SO2-.(formed by the predissociation of dithionite ion) with k = 3 X 10(6) M-1.s-1. This rate constant is at least 30 times lower than that for the reduction of free Kp2ox (k greater than 10(8) M-1.s-1). Two mechanisms have been considered and limits obtained for the rate constants for MgADP binding/dissociation and a protein conformation change. Both mechanisms give rate constants (e.g. MgADP binding 3 X 10(5) less than k less than 3 X 10(6) M-1.s-1 and protein conformation change 6 X 10(2) less than k less than 6 X 10(3) s-1) that are similar to those reported for creatine kinase (EC 2.7.3.2). The kinetics also show that in the catalytic cycle of nitrogenase with sodium dithionite as reductant replacement of 2MgADP by 2MgATP occurs on reduced and not oxidized Kp2. Although the Kp2ox was reduced stoichiometrically by SO2-. and bound two equivalents of MgADP with complete conversion into the less-reactive conformation, it was only 45% active with respect to its ability to effect MgATP-dependent electron transfer to the MoFe protein.     

Rapid spectral scan and stopped-flow studies of carbon monoxide binding to bovine adrenocortical cytochrome P-450scc

Carbon monoxide binding with both cholesterol-free (low-spin) and cholesterol-bound (high-spin) reduced forms of purified cytochrome P-450scc has been investigated by rapid-scan and stopped-flow spectrometry. CO binding occurs within 150 ms at 25 degrees C for both forms of P-450scc, with a typical absorption maximum at 450 nm. Isosbestic points occur at the following wavelengths: between reduced-CO and reduced cholesterol-free P-450scc at 434 and 471 nm; between reduced-CO and reduced cholesterol-bound P-450scc at 433 and 469 nm. Both the 'on' (k1) and 'off' rate constants (k-1) are found to be independent of pH between pH 5 and 9. The mean values of k1 for cholesterol-free (1.8 +/- 0.2) X 10(5) M-1 X s-1) and cholesterol-bound [1.9 +/- 0.1) X 10(5) M-1 X s-1) P-450scc are almost identical, while the mean value of k-1 for the former [2.3 +/- 0.3) X 10 s-1) is about double that of the latter [1.2 +/- 0.1) X 10 s-1). This suggests the instability of the reduced-CO complex in the absence of cholesterol.     

Kinetics and mechanism of the reduction of horse heart ferricytochrome c by glutathione.

A detailed investigation of the reduction of cytochrome c by glutathione has shown that the reaction proceeds through several steps. A rapid combination of the reducing agent with the cytochrome leads to the formation of a glutathione-cytochrome intermediate in which the glutathione most likely interacts with the edge of the heme moiety. The electron transfer takes place in a subsequent slower step. Since cytochrome c(III) exists in two conformational forms at neutral pH [Kujundzic, N., & Everse, J. (1978) Biochem. Biophys. Res. Commun. 82, 1211], the reduction of cytochrome c by glutathione may be represented by cyt c(III) + GS- reversible K1 cyt c(III) ... GS- reversible k1 products cyt c*(III) + GS- reversible K2 cyt c*(III) ... GS- reversible k2 products At 25 degrees C, pH 7.5, and an ionic strength of 1.0 (NaCl), k1 = 1.2 X 10(-3) S-1, k2 = 2.0 X 10(-3) S-1, k1 = 2.9 X 10(3) M-1, and K2 = 5.3 X 10(3) M-1. The reaction is catalyzed by trisulfides, and second-order rate constants of 4.55 X 10(3) and 7.14 X 10(3) M-1 S-1 were obtained for methyl trisulfide and cysteine trisulfide, respectively.     

Static and kinetic studies on carp muscle parvalbumins

Fluorescence titration and fluorescence stopped-flow studies were performed on carp muscle parvalbumin components 1, 2, 3, and 5 (the latter three components were modified with a SH-directed fluorescent reagent, dansyl-L-cysteine). Apparent binding constants (Kapp) of Ca2+ to these components decrease in the order of component 2 (Kapp = 2.8 +/- 0.9 X 10(8) M-1) greater than component 1 (Kapp = 1.25 +/- 0.25 X 10(8) M-1) greater than component 3 = component 5 (Kapp = 4.0 +/- 0.5 X 10(7) M-1) in 30 mM KCl, 50 mM Na-cacodylate-HCl, pH 7.0 at 20 degrees C. The rate constant of the conformational change of parvalbumin induced by Ca2+ binding or removal decreases in the order of component 2 greater than component 1 greater than component 5 greater than or equal to component 3; that is, component 2 undergoes the fastest conformational change and component 3 the slowest in response to the rapid free Ca2+ concentration ([Ca2+]) change in the protein solution. The fluorescence titration curves and [Ca2+]-dependences of the rate constants are analyzed by a simple two-state model, (partially unfolded state) k1 in equilibrium k2 (folded state). It is shown that the equilibrium constant K = k1/k2 depends on the second power of [Ca2+], the rate constant k1 on the first power of [Ca2+] and k2 on the inverse first power of [Ca2+], respectively.     

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.     

Purification, characterization and ion binding properties of human brain S100b protein

Human brain S100b (beta beta) protein was purified using zinc-dependent affinity chromatography on phenyl-Sepharose. The calcium- and zinc-binding properties of the protein were studied by flow dialysis technique and the protein conformation both in the metal-free form and in the presence of Ca2+ or Zn2+ was investigated with ultraviolet spectroscopy, sulfhydryl reactivity and interaction with a hydrophobic fluorescence probe 6-(p-toluidino)naphthalene-2-sulfonic acid (TNS). Flow dialysis measurements of Ca2+ binding to human brain S100b (beta beta) protein revealed six Ca2+-binding sites which we assumed to represent three for each beta monomer, characterized by the macroscopic association constants K1 = 0.44 X 10(5) M-1; K2 = 0.1 X 10(5) M-1 and K3 = 0.08 X 10(5) M-1. In the presence of 120 mM KCl, the affinity of the protein for calcium is drastically reduced. Zinc-binding studies on human S100b protein showed that the protein bound two zinc ions per beta monomer, with macroscopic constants K1 = 4.47 X 10(7) M-1 and K2 = 0.1 X 10(7) M-1. Most of the Zn2+-induced conformational changes occurred after the binding of two zinc ions per mole of S100b protein. These results differ significantly from those for bovine protein and cast doubt on the conservation of the S100 structure during evolution. When calcium binding was studied in the presence of zinc, we noted an increase in the affinity of the protein for calcium, K1 = 4.4 X 10(5) M-1; K2 = 0.57 X 10(5) M-1; K3 = 0.023 X 10(5) M-1. These results indicated that the Ca2+- and Zn2+-binding sites on S100b protein are different and suggest that Zn2+ may regulate Ca2+ binding by increasing the affinity of the protein for calcium.     

[Nitrotyrosyl]cytochrome c. Studies of the effect of iron binding, protein denaturants and oxidation-reduction potentials.

Static measurements of the reaction of ligand binding were done by conventional spectrophotometry. The ligand-binding reactions with nitrated cytochrome c were performed with imidazole, iminazole, CO and NO. The stoicheiometry was found to be 1:1, and the stability constants for the complexes formed between the nitrated cytochrome c and the ligands are: 2.58 X 10(4) M-1 (imidazole); 1.01 X 10(2) M-1 (iminazole); 3.6 X 10(4) M-1 (CO); 2.74 X 10(4) M-1 (NO). It was found that the electrometric potentials at pH 7.0 and 25degreesC of [aminotyrosyl]cytochrome c are E'o form II = 0.115 V and E'o form I = 0.260 V, where forms I and II are two species of protein co-existing in the protein solution. The isoelectric point for the oxidized form of [nitrotyrosyl]cytochrome c was 10.05, at 4degreesC.     

Laser-flash-photolysis studies of p-cresol methylhydroxylase. Electron-transfer properties of the flavin and haem components.

p-Cresol methylhydroxylase, a heterodimer consisting of one flavoprotein subunit and one cytochrome c subunit, may be resolved into its subunits, and the holoenzyme may then be fully reconstituted from the pure subunits. In the present study we have characterized the reduction kinetics of the intact enzyme and its subunits, by using exogenous 5-deazariboflavin semiquinone radical generated in the presence of EDTA by the laser-flash-photolysis technique. Under anaerobic conditions the 5-deazariboflavin semiquinone radical reacts rapidly with the native enzyme with a rate constant approaching that of a diffusion-controlled reaction (k = 2.8 X 10(9) M-1 X s-1). Time-resolved difference spectra at pH 7.6 indicate that both flavin and haem are reduced initially by the deazariboflavin semiquinone radical, followed by an additional slower intramolecular electron transfer (k = 220 s-1) from the endogenous neutral flavin semiquinone radical to the oxidized haem moiety of the native enzyme. During the steady-state photochemical titration of the native enzyme at pH 7.6 with deazariboflavin semiquinone radical generated by light-irradiation the haem appeared to be reduced before the protein-bound flavin and was followed by the formation of the protein-bound anionic flavin radical. This result suggests that the redox potential of the haem is higher than that of the flavin, and that deprotonation of the flavin neutral radical occurred during the photochemical titration. Reduction kinetics of the flavoprotein and cytochrome subunits were also investigated by laser-flash photolysis. The protein-bound flavin of the isolated flavin subunit was reduced rapidly by the deazariboflavin semiquinone radical (k = 2.2 X 10(9) M-1 X s-1), as was the haem of the pure cytochrome c subunit (k = 3.7 X 10(9) M-1 X s-1). Flash-induced difference spectra obtained for the flavoprotein and cytochrome subunits at pH 7.6 were consistent with the formation of neutral flavin semiquinone radical and reduced haem, respectively. Investigation of the kinetic properties of the neutral flavin semiquinone radical of the flavoprotein subunit at pH 7.6 and at longer times (up to 5s) were consistent with a slow first-order deprotonation reaction (k = 1 s-1) of the neutral radical to its anionic form.     

Ligand binding equilibrium and kinetic measurements on the dimeric myoglobin of Busycon canaliculatum and the comparative ligand binding of diverse non-cooperative heme proteins

The rate constants and delta H degrees for the non-cooperative dimeric Busycon myoglobin are: oxygen, k' = 4.75 X 10(7) M-1 sec-1, k = 71 sec-1, and CO, l'= 3.46 X 10(5) M-1 sec-1, l = 0.0052 sec-1 at 20 degrees C, pH 7, delta H degrees = -3 kcal/mol for O2 and CO.2. Log-log plots of k vs K for oxygen and of l' vs L for CO binding for numerous non-cooperative hemoglobins and myoglobins point to a large steric influence of the protein on heme ligation reactions. Many of the proteins behave as "R" state for one ligand, but "T" for the other.     

Electron transfer from cytochrome b5 to iron and copper complexes

The rates of electron transfer from the tryptic fragment of bovine liver cytochrome b5 to FeIIINTA, FeIIIATP, CuIINTA, CuIIATP, and CuIIHis have been measured by anaerobic stopped-flow techniques. The rates of reduction of the Fe(III) complexes are independent of ionic strength, enhanced at low pH, and slightly inhibited by ZnIINTA. Saturation kinetics are observed with CuIINTA (kappa et = 0.05 s-1, K = 8.6 M-1), CuIIHis (kappa et = 0.2 s-1, K = 2.6 X 10(3) M-1), and CuIIATP (kappa et = 0.6 s-1, K = 4.5 X 10(3) M-1), thereby indicating that binding of Cu(II) to the protein occurs prior to electron transfer. 1H NMR resonances of the three surface histidines and some neighboring residues have been assigned by two-dimensional NMR techniques. NMR titration experiments show unequivocally that CuIINTA binds preferentially at a site near His-26 and Tyr-27.     

The reaction between cytochrome c1 and cytochrome c

The kinetics of electron transfer between the isolated enzymes of cytochrome c1 and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c1 and ferricytochrome c is fast; the second-order rate constant (k1) is 3.0 . 10(7) M-1 . s-1 at low ionic strength (I = 223 mM, 10 degrees C). The value of this rate constant decreases to 1.8 . 10(5) M-1 . s-1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c1 and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c1 and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c1 and ferricytochrome c and the reverse reaction are k1 = 2.4 . 10(5) M-1 . s-1 and k-1 = 3.3 . 10(5) M-1 . s-1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10 degrees C). The 'equilibrium' constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c2+1 + cytochrome c3+ in equilibrium or formed from cytochrome c3+1 + cytochrome c2+.     

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.