The oxidation of Pseudomonas cytochrome c-551 oxidase by potassium ferricyanide.

Stopped-flow kinetics were made of the reaction between ascorbate-reduced Pseudomonas cytochrome oxidase and potassium ferricyanide under both N2 and CO atmospheres. Under N2 three kinetic processes were observed, two being dependent on ferricyanide concentration, with second-order rate constants of 9.6 X 10(4)M-1.s-1 and 1.5 X 10(4)M-1.s-1, whereas the other was concentration-independent, with a first-order rate constant of 0.17 +/- 0.03s-1. Measurements of their kinetic difference spectra have allowed the fastest and second-fastest phases of the reaction to be assigned to direct bimolecular reactions of ferricyanide with the haem c and haem d, moieties of the enzyme respectively. Under CO, the second-order rate constant for the reaction of the haem c was, at 1.3 X 10(5)M-1.s-1, slightly enhanced over the rate in a N2 atmosphere, but the reaction velocity of the haem d1 component was greatly decreased, being apparently limited to that of the rates of CO dissociation from the molecule (0.15s-1 and 0.03s-1). The results are compared with those obtained during a previous study of the reaction of reduced Pseudomonas cytochrome oxidase with oxidized azurin.     

The reaction of Pseudomonas aeruginosa cytochrome c-551 oxidase with oxygen.

The reaction of ascorbate-reduced Pseudomonas cytochrome oxidase with oxygen was studied by using stopped-flow techniques at pH 7.0 and 25 degrees C. The observed time courses were complex, the reaction consisting of three phases. Of these, only the fastest process, with a second-order rate constant of 3.3 X 10(4) M-1.S-1, was dependent on oxygen concentration. The two slower processes were first-order reactions with rates of 1.0 +/- 0.4s-1 and 0.1 +/- 0.03s-1. A kinetic titration experiment revealed that the enzyme had a relatively low affinity constant for oxygen, approx. 10(4)M-1. Kinetic difference spectra were determined for all three reaction phases, showing each to have different characteristics. The fast-phase difference spectrum showed that changes occurred at both the haem c and haem d1 components of the enzyme during this process. These changes were consistent with the haem c becoming oxidized, but with the haem d1 assuming a form that did not correspond to the normal oxidized state, a situation that was not restored even after the second kinetic phase, which reflected further changes in the haem d1 component. The results are discussed in terms of a kinetic scheme.     

The kinetics of electron transfer between pseudomonas aeruginosa cytochrome c-551 and its oxidase.

The redox reaction between cytochrome c-551 and its oxidase from the respiratory chain of pseudomonas aeruginosa was studied by rapid-mixing techniques at both pH7 and 9.1. The electron transfer in the direction of cytochrome c-551 reduction, starting with the oxidase in the reduced and CO-bound form, is monophasic, and the governing bimolecular rate constants are 1.3(+/- 0.2) x 10(7) M-1 . s-1 at pH 9.1 and 4 (+/- 1) x 10(6) M-1 . s-1 at pH 7.0. In the opposite direction, i.e. mixing the oxidized oxidase with the reduced cytochrome c-551 in the absence of O2, both a lower absorbance change and a more complex kinetic pattern were observed. With oxidized azurin instead of oxidized cytochrome c-551 the oxidation of the c haem in the CO-bound oxidase is also monophasic, and the second-order rate constant is 2 (+/- 0.7) x 10(6) M-1 . s-1 at pH 9.1. The redox potential of the c haem in the oxidase, as obtained from kinetic titrations of the completely oxidized enzyme with reduced azurin as the variable substrate, is 288 mV at pH 7.0 and 255 mV at pH 9.1. This is in contrast with the very high affinity observed in similar titrations performed with both oxidized azurin and oxidized cytochrome c-551 starting from the CO derivative of the reduced oxidase. It is concluded that: (i) azurin and cytochrome c-551 are not equally efficient in vitro as reducing substrates of the oxidase in the respiratory chain of Pseudomonas aeruginosa; (ii) CO ligation to the d1 haem in the oxidase induces a large decrease (at least 80 mV) in the redox potential of the c-haem moiety.     

A re-examination of the reactions of cyanide with cytochrome c oxidase

Experiments were performed to examine the cyanide-binding properties of resting and pulsed cytochrome c oxidase in both their stable and transient turnover states. Inhibition of the oxidation of ferrocytochrome c was monitored as a function of cyanide concentration. Cyanide binding to partially reduced forms produced by mixing cytochrome c oxidase with sodium dithionite was also examined. A model is presented that accounts fully for cyanide inhibition of the enzyme, the essential feature of which is the rapid, tight, binding of cyanide to transient, partially reduced, forms of the enzyme populated during turnover. Computer fitting of the experimentally obtained data to the kinetic predictions given by this model indicate that the cyanide-sensitive form of the enzyme binds the ligand with combination constants in excess of 10(6) M-1 X s-1 and with KD values of 50 nM or less. Kinetic difference spectra indicate that cyanide binds to oxidized cytochrome a33+ and that this occurs rapidly only when cytochrome a and CuA are reduced.     

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.     

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.     

Cytochrome c peroxidase activity of a protease-modified form of cytochrome c-552 from the denitrifying bacterium Pseudomonas perfectomarina

Protease activity present in aerobically grown cells of Pseudomonas perfectomarina, protease apparently copurified with cytochrome c-552, and trypsin achieved a limited proteolysis of the diheme cytochrome c-552. That partial lysis conferred cytochrome c peroxidase activity upon cytochrome c-552. The removal of a 4000-Da peptide explains the structural changes in the cytochrome c-552 molecule that resulted in the appearance of both cytochrome c peroxidase activity (with optimum activity at pH 8.6) and a high-spin heme iron. The oxidized form of the modified cytochrome c-552 bound cyanide to the high-spin ferric heme with a rate constant of (2.1 +/- 0.1) X 10(3) M-1 s-1. The dissociation constant was 11.2 microM. Whereas the intact cytochrome c-552 molecule can be half-reduced by ascorbate, the cytochrome c peroxidase was not reducible by ascorbate, NADH, ferrocyanide, or reduced azurin. Dithionite reduced the intact protein completely but only half-reduced the modified form. The apparent second-order rate constant for dithionite reduction was (7.1 +/- 0.1) X 10(2) M-1 s-1 for the intact protein and (2.2 +/- 0.1) X 10(3) M-1 s-1 for the modified form. In contrast with other diheme cytochrome c peroxidases, reduction of the low-spin heme was not necessary to permit ligand binding by the high-spin heme iron.     

An investigation of the ligand-binding properties of Pseudomonas aeruginosa nitrite reductase.

The low-temperature e.p.r. and m.c.d. (magnetic-circular-dichroism) spectra of Pseudomonas aeruginosa nitrite reductase, together with those of its partially and fully cyanide-bound derivatives, were investigated. The m.c.d. spectra in the range 600-2000 nm indicate that the native axial ligands to haem c are histidine and methionine, and furthermore that it is the methionine ligand that must be displaced before cyanide binding at this haem. The m.c.d. spectra in the range 1000-2000 nm contain no charge-transfer bands arising from low-spin ferric haem d1, a chlorin. New optical transitions in the region 700-850 nm were found for the cyanide adduct of haem d1. The g-values of haem d1 in the native enzyme are 2.51, 2.43 and 1.71, suggesting co-ordination by two histidine ligands in the oxidized state. There is clear evidence in the e.p.r. data of an interaction between the c and d1 haem groups. This is not apparent in the optical spectra. The results are interpreted in terms of haem groups that are remote from each other, their interaction being mediated through protein conformational changes. The possible implications of this in relation to reduction processes catalysed by the enzyme are considered.     

Nickel-specific, slow-binding inhibition of carbon monoxide dehydrogenase from Rhodospirillum rubrum by cyanide

The inhibition of purified carbon monoxide dehydrogenase from Rhodospirillum rubrum by cyanide was investigated in both the presence and absence of CO and electron acceptor. The inhibition was a time-dependent process exhibiting pseudo-first-order kinetics under both sets of conditions. The true second-order rate constants for inhibition were 72.2 M-1 s-1 with both substrates present and 48.9 and 79.5 M-1 s-1, respectively, for the reduced and oxidized enzymes incubated with cyanide. CO partially protected the enzyme against inhibition after 25-min incubation with 100 microM KCN. Dissociation constants of 8.46 microM (KCN) and 4.70 microM (CO) were calculated for the binding of cyanide and CO to the enzyme. Cyanide inhibition was fully reversible under an atmosphere of CO after removal of unbound cyanide. N2 was unable to reverse the inhibition. The competence of nickel-deficient (apo) CO dehydrogenase to undergo activation by NiCl2 was unaffected by prior incubation with cyanide. Cyanide inhibition of holo-CO dehydrogenase was not reversed by addition of NiCl2. 14CN- remained associated with holoenzyme but not with apoenzyme through gel filtration chromatography. These findings suggest that cyanide is a slow-binding, active-site-directed, nickel-specific, reversible inhibitor of CO dehydrogenase. We propose that cyanide inhibits CO dehydrogenase by being an analogue of CO and by binding through enzyme-bound nickel.     

Some magnetic properties of Pseudomonas cytochrome oxidase.

The magnetic properties of the haem groups of Pseudomonas cytochrome oxidase and its cyanide-bound derivatives were studied in both the oxidized and reduced states by means of m.c.d. (magnetic circular dichroism) at low temperatures. In addition, the oxidized forms of the enzyme were also investigated by e.p.r. (electron-paramagnetic-resonance) spectroscopy, and a parallel study, using both e.p.r. and m.c.d., was made on Pseudomonas cytochrome c-551 to aid spectral assignments. For ascorbate-reduced Pseudomonas cytochrome oxidase, the temperature-independence of those features in the m.c.d. spectrum corresponding to the haem c, and the temperature-dependence of those signals corresponding to the haem d1, showed the former to be low-spin and the latter to be high-spin (s = 2). However, addition of cyanide to the reduced enzyme gave a form of the protein that was completely low-spin. The e.p.r. and m.c.d. sectra of oxidized Pseudomonas cytochrome oxidase and its cyanide derivative were consistent with the haem c and d1 components being low-spin in both cases. Pseudomonas cytochrome c-551 was found to be low-spin in both its oxidized and reduced redox states.     

Cyanide binding to canine myeloperoxidase

Equilibria and kinetics of cyanide binding to canine myeloperoxidase were studied. Spectral results support the presence of two heme binding sites; an isosbestic point at 444 nm and a linear Scatchard plot suggest that the binding affinity of cyanide to the two subunits of the enzyme is the same. The dissociation constant is 0.53 microM. The pH dependence of the apparent second order rate constant indicates the presence of an acid-base group on the enzyme with a pKa of 3.8 +/- 0.1. The protonated form of cyanide binds to the basic enzyme with a rate constant of (4.3 +/- 0.3) x 10(6) M-1 s-1.     

Rates of cyanide binding to the catalytic intermediates of mammalian cytochrome c oxidase, and the effects of cytochrome c and poly(L-lysine).

Rate constants of cyanide binding to 'fast' oxidase have been measured in the fully-oxidised (O), peroxy (P) and ferryl (F) states at pH 8.0. Values of 2.2, 8 and 10 M-1 s-1, respectively, were obtained. Thus, none of these states appears to exhibit a rate that would identify it as the species responsible for the extremely rapid cyanide binding observed during turnover. On the other hand, with 'oxidised' enzyme as prepared, containing a very small fraction of one-electron-reduced (E state) oxidase, a corresponding fraction of enzyme exhibited spectral changes consistent with cyanide binding with a rate constant in excess of 10(4) M-1 s-1. Evidence is presented suggesting that mediation of electron transfer from one-electron-reduced, cyanide-liganded enzyme to free, ferric oxidase, rather than a global protein conformational change of the enzyme, is responsible for the greatly enhanced cyanide binding rates seen in the presence of cytochrome c or poly(L-lysine). Inter-oxidase electron exchange in 'oxidised' enzyme can result in a complicated dependence of the binding rate on cyanide concentration. We have demonstrated that this may give rise to a saturation of the rate of cyanide binding.     

The reaction mechanism of the novel vanadium-bromoperoxidase. A steady-state kinetic analysis

The reaction of vanadium-bromoperoxidase from the brown alga Ascophyllum nodosum with hydrogen peroxide, bromide, and 2-chlorodimedone has been subjected to an extensive steady-state kinetic analysis. Systematic variation of pH and the concentrations of these three components demonstrate that the reaction model includes four enzyme species: native bromoperoxidase, a bromoperoxidase-bromide inhibitory complex, a bromoperoxidase-hydrogen peroxide intermediate, and a bromoperoxidase-HOBr species. This latter intermediate did not display any direct interaction with the nucleophilic reagent as oxidized bromine species (Br-3, Br2, and/or HOBr) were the primary reaction products. The generation of oxidized bromine species was as fast as the bromination of 2-chlorodimedone. The enzyme did not show any specificity with regard to bromination of various organic compounds. Formation of the bromoperoxidase-bromide inhibitory complex was competitive with the reaction between hydrogen peroxide and enzyme. From the steady-state kinetic data lower limits for the second-order rate constants at various pH values were calculated for individual steps in the catalytic cycle. This pH study showed that native enzyme must be unprotonated prior to binding of hydrogen peroxide (second-order association rate constant of 2.5.10(6) M-1.s-1 at pH greater than 6). The pKa for the functional group controlling the binding of hydrogen peroxide was 5.7 and is ascribed to a histidine residue. The reaction rate between bromide and enzyme-hydrogen peroxide intermediate also depended on pH (second-order association rate constant of 1.7.10(5) M-1.s-1 at pH 4.0).     

Laser flash photolysis studies of the reduction kinetics of NADPH:cytochrome P-450 reductase

The reduction kinetics of NADPH:cytochrome P-450 reductase have been investigated by the laser flash photolysis technique, using the semiquinone of 5-deazariboflavin (5-dRfH.) as the reductant. Transients observed at 470 nm at neutral pH indicated that the oxidized reductase was reduced via second-order kinetics with a rate constant of 6.8 X 10(7) M-1 s-1. The second-order rate constant corresponding to the formation of the protein-bound semiquinone (measured at 585 nm) was essentially the same as that obtained at 470 nm (7.1 X 10(7) M-1 s-1). Subsequent to this rapid formation of protein-bound semiquinone, a partial exponential decay was observed at 585 nm. The rate of this decay remained invariant with protein concentration between pH 5.0 and 7.0, and a first-order rate constant of 70 s-1 was obtained for this process. This is assigned to intramolecular electron transfer from FADH. to FMN. Prior reduction of the enzyme to the one-electron level led to a decrease in both the second-order rate constant for reduction (2 X 10(7) M-1 s-1) and the first-order intraflavin electron transfer rate constant (15 s-1). The protein-bound FAD moiety of FMN-depleted reductase was reduced by 5-dRfH. with a second-order rate constant that was identical with that observed with the native enzyme (6.9 X 10(7) M-1 s-1). However, with this species no significant decay of the FAD semiquinone was observed at 585 nm following its rapid formation, consistent with the above assignment of this kinetic process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of photosynthetic electron transfer in artificial vesicles reconstituted with purified complexes from Rhodobacter capsulatus. I. The interaction of cytochrome c2 with the reaction center

1. The kinetics of the interaction of cytochrome c2 and photosynthetic reaction centers purified from Rhodobacter capsulatus were studied in proteoliposomes reconstituted with a mixture of phospholipids simulating the native membrane (i.e. containing 25% L-alpha-phosphatidylglycerol). 2. At low ionic strength, the kinetics of cytochrome-c2 oxidation induced by a single turnover flash was very different, depending on the concentration of cytochrome c2: at concentrations lower than 1 microM, the process was strictly bimolecular (second-order rate constant, k = 1.7 x 10(9) M-1 s-1), while at higher concentrations a fast oxidation process (half-time lower than 20 microseconds) became increasingly dominant and encompassed the total process at a cytochrome c2 concentration around 10 microM. From the concentration dependence of the amplitude of this fast phase an association constant for a reaction-center--cytochrome-c2 complex of about 10(5) M-1 was evaluated. From the fraction of photo-oxidized reaction centers promptly re-reduced in the presence of saturating concentrations of externally added cytochrome c2, it was found that in approximately 60% of the centers the cytochrome-c2 site was exposed to the external compartment. 3. Both the second-order oxidation reaction and the formation of the reaction-center--cytochrome-c2 complex were very sensitive to ionic strength. In the presence of 180 mM KCl, the value of the second-order rate constant was decreased to 7.0 x 10(7) M-1 s-1 and no fast oxidation of cytochrome c2 could be observed at 10 microM cytochrome c2. 4. The kinetics of exchange of oxidized cytochrome c2 bound to the reaction center with the reduced form of the same carrier, following a single turnover flash, was studied in double-flash experiments, varying the dark time between photoactivations over the range 30 microseconds to 5ms. The experimental results were analyzed according to aminimal kinetic model relating the amounts of oxidized cytochrome c2 and reaction centers observable after the second flash to the dark time between flashes. This model included the rate constants for the electron transfer between the primary and secondary ubiquinone acceptors of the complex (k1) and for the exchange of cytochrome c2 (k2). Fitting to the experimental results indicated a value of k1 equal to 2.4 x 10(3) s-1 and a lower limit for k2 of approximately 2 x 10(4) s-1 (corresponding to a second-order rate constant of approximately 3 x 10(9) M-1 s-1).     

Internal electron transfer and structural dynamics of cd1 nitrite reductase revealed by laser CO photodissociation.

Laser photolysis techniques have been employed to investigate the internal electron transfer (eT) reaction within Pseudomonas aeruginosa nitrite reductase (Pa-NiR). We have measured the (d1--> c) internal eT rate for the wild-type protein and a site-directed mutant (Pa-NiR H327A) which has a substitution in the d1-heme binding pocket; we found the rate of eT to be fast, keT = 2.5 x 10(4) and 3.5 x 10(4) s-1 for the wild-type and mutant Pa-NiR, respectively. We also investigated the photodissociation of CO from the fully reduced proteins and observed microsecond first-order relaxations; these imply that upon breakage of the Fe2+-CO bond, both Pa-NiR and Pa-NiR H327A populate a nonequilibrium state which decays to the ground state with a complex time course that may be described by two exponential processes (k1 = 3 x 10(4) s-1 and k2 = 0.25 x 10(4) s-1). These relaxations do not have a kinetic difference spectrum characteristic of CO recombination, and therefore we conclude that Pa-NiR undergoes structural rearrangements upon dissociation of CO. The bimolecular rate of CO rebinding is 5 times faster in Pa-NiR H327A than in the wild-type enzyme (1.1 x 10(5) M-1 s-1 compared to 2 x 10(4) M-1 s-1), indicating that this mutation in the active site alters the CO diffusion properties of the protein, probably reducing steric hindrance. CO rebinding to the wild-type mixed valence enzyme (c3+d12+) which is very slow (k = 0.25 s-1) is proposed to be rate-limited by the c --> d1 internal eT event, involving the oxidized d1-heme which has a structure characteristic of the fully oxidized and partially oxidized Pa-NiR.     

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.     

Kinetics and mechanism of anionic ligand binding to carbonic anhydrase

The kinetics of complex formation between Co(II)-carbonic anhydrase B and the anions cyanate, thiocyanate and cyanide has been studied at different pH values employing temperature-jump relaxation spectrometry. Formation of the 1:1 complex occurs via binding of the deprotonated state of the anion to an acidic state of the enzyme. The determined formation rate constants range from 10(8) to 3 X 10(9) M-1 s-1 and are two to three orders of magnitude higher than the value estimated for a ligand coordination to the central Co2+, based on a solvate substitution mechanism. These kinetic results strongly indicate that the deprotonated anion binds to an unoccupied coordination position of the protein-bound heavy metal ion in the form of an addition reaction. Upon binding of the anion, the coordination number of the Co2+ in the acidic state of the enzyme is increased from four to five. In the case of cyanide, a 2:1 anion complex is also formed. The formation rate constant is 5 X 10(5) M-1 s-1 which provides good evidence that this binding process is controlled by a solvate substitution mechanism.     

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 [methionine sulphoxide] cytochrome c.

A cytochrome c haem ligand, methionine-80, was photo-oxidized to methionine sulphoxide and the subsequent changes in redox properties and ligand binding were monitored kinetically. Isoelectric focusing of the product showed the presence of a single oxidized species, capable of binding CO when reduced. The binding of CO to the reduced protein was followed in stopped-flow experiments, which revealed the presence of two binding processes, at neutral pH, with rate constants of K+1 = 3.4 X 10(3)M-1-S-1 and k+2 = 5.80 X 10(2)M-1-S-1. When CO was photolytically dissociated from the reduced protein two recombination processes were observed with rates almost identical with those observed in the stopped-flow experiments (k+1 = 3.3 X 10(3)M-1-S-1 and k+2 = 6.0 X 10(2)M-1-S-1). These findings provide evidence of two reduced forms of the protein. The reduction of [methionine sulphoxide]cytochrome c by Cr2+ at neutral pH in stopped-flow experiments showed the presence of a single second-order reduction process (k = 7.2 X 10(3)M-1-S-1, activation energy = 44kJ/mol) and one first-order process. This protein was compared with some other chemically modified cytochromes.