Reduction of laccase type 1 copper by 3,4-dihydroxyphenylalanine and other catechol derivatives

3,4-Dihydroxyphenylalanine (DOPA) is not a preferred substrate of Rhus vernicifera laccase, as rate constants for the anaerobic reduction of the type 1 cupric atom by L-DOPA (6.3 X 10(1) M-1 s-1), D-DOPA (2.6 X 10(1) M-1 s-1), and L-DOPA methyl ester (2.6 X 10(1) M-1 s-1) are considerably smaller than k1 (catechol) (7 X 10(2) M-1 s-1) and rate constants characteristic of numerous other nonphysiological organic substrates (25 degrees C, pH 7.0, I = 0.5 M). The reactions of DOPA derivatives with laccase are unique, however, in that a two-term rate law pertains: kobsd = k0 + k1[phenol]; k0(L-DOPA) = 7 X 10(-2) s-1. The reactivities of other catechol derivatives (pyrogallol, gallic acid, and methyl gallate) with laccase type 1 copper were also examined.     

Kinetics of electron transfer between mitochondrial cytochrome c and iron hexacyanides

The reduction of horse and Candida krusei cytochromes c by ferrocyanide has been studied by 1H NMR spectroscopy and the reaction found to involve a precursor complex of ferrocyanide bound to ferricytochrome c (pH* 7.4, 2H2O, I = 0.12, and 25 degrees C). The electron transfer rate constants for the reduction of the two ferricytochromes by associated ferrocyanide were found to be the same at 780 +/- 80 sec-1 but the association constants for binding of ferrocyanide to ferricytochrome c were significantly different: horse, 90 +/- 20 M-1 and Candida, 285 +/- 30 M-1. The different association constants partly accounts for the previously observed reactivity difference between horse and Candida cytochromes c. Comparison of the NMR data with data obtained by other kinetic methods has allowed the electron transfer rate constant for the oxidation of ferrocytochrome c by associated ferricyanide to be determined. This was found to be 4.6 +/- 1 X 10(4) sec-1.     

Direct measurement of intramolecular electron transfer between type I and type III copper centers in the multi-copper enzyme ascorbate oxidase and its type II copper-depleted and cyanide-inhibited forms

Transient kinetics of reduction of zucchini squash ascorbate oxidase (AO) by lumiflavin semiquinone have been studied by using laser flash photolysis. Second-order kinetics were obtained for reduction of the type I copper with a rate constant of 2.7 X 10(7) M-1 s-1, which is comparable to that obtained with other blue copper proteins such as plastocyanin. Following reduction, the type I copper was reoxidized in a protein concentration independent (i.e., intramolecular) reaction (kobs = 160 s-1). Comparison with literature values for limiting rate constants in transient single-turnover kinetic experiments suggests that intramolecular electron transfer probably is the rate-limiting step in enzyme catalysis. The extent of reoxidation of type I copper was approximately 55%, which is consistent with the approximately equal redox potentials of the type I and type III copper centers. Neither azide nor fluoride caused any significant changes in kinetics, although they are enzyme inhibitors and are thought to bind to the type II copper. In contrast, cyanide caused a concentration-dependent decrease in the extent of intramolecular electron transfer (with no change in rate constant), and decreased the rate constant for reduction of the type I copper by a factor of 2. The apparent dissociation constant for cyanide (0.2-0.4 mM) is similar to that reported for inhibition of enzyme activity. Removal of the type II copper from AO only marginally affected the kinetics of electron transfer to type I copper (k = 3.2 x 10(7) M-1 s-1) and slightly increased the extent but did not alter the rate constant of intramolecular electron transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Kinetics and mechanism of Zn(II) complexation with reduced glutathione

The kinetics of formation and dissociation of mono and bis complexes of Zn(II) with reduced glutathione (H4L+ = fully protonated form) were studied in aqueous solution at 25.0 +/- 0.1 degrees C and ionic strength 0.30 M (NaNO3) in the pH range 4.58 to 4.98 by temperature-jump. The reaction was found to proceed via two different mechanisms depending on degree of ligand protonation. In both cases, complex formation is predominantly if not completely through the sulfur. Reaction with the form HL-2 (only the amino nitrogen protonated), the dominant form of this species, proceeds by the expected rat limiting water loss (dissociative or Eigen) mechanism with rate constants of 9.3 X 10(7) M-1 sec-1 (+/- 24%) for mono and 5.1 X 10(7) M-1 sec-1 (+/- 25%) for bis complex formation. Reaction with H2L--(sulfur protonated) yields rate constants of 3.9 X 10(3) M-1 sec-1 (+/- 43%) for mono and 1.95 X 10(3) M-1 sec-1 (+/- 43%) for bis complex formation. The decrease in rate constant is attributed to blockage of the complexing site on reduced glutathione by intramolecular hydrogen bonding, with proton removal being the rate determining step.     

The modulation of cytochrome c electron self-exchange by site-specific chemical modification and anion binding

The site-specific chemical modification of horse heart cytochrome c at Lys-13 and -72 using 4-chloro-3,5-dinitrobenzoic acid (CDNB) increases the electron self-exchange rate of the protein. In the presence of 0.24 M cacodylate (pH* 7.0) the electron self-exchange rate constants, kex, measured by a 1H NMR saturation transfer method at 300 K, are 600, 6 X 10(3) and 6 X 10(4) M-1 X s-1 for native, CDNP-K13 and CDNP-K72 cytochromes c respectively. Repulsive electrostatic interactions, which inhibit cytochrome c electron self-exchange, are differentially affected by modification. Measurements of 1H NMR line broadening observed with partially oxidised samples of native cytochrome c show that ATP and the redox inert multivalent anion Co(CN)3-6 catalyse electron self-exchange. At saturation a limiting value of approximately 1.4 X 10(5) M-1 X s-1 is observed for both anions.     

Kinetics of the oxidation of Rhus vernicifera stellacyanin by the Co(EDTA)-- ion.

A kinetic study of the oxidation of the copper(I) form of the blue copper protein stellacyanin (St(I) by Co(EDTA)-- has been performed. Observed rate constants approach a saturation limit with increasing [Co(EDTA)--] at pH 7, consistent with a mechanism involving rapid pre-equilibrium oxidant-protein complex formation followed by rate-limiting intramolecular Cu(I) to Co(III) electron transfer: Co(EDTA)-- + St(i Qp in equilibrium Co(EDTA)-- ---St(I) Co(EDTA)-- ---St(I) k2 leads to Co(EDTA)2-- ---St(II) (Qp = 149 M--1, k2 = 0.169 sec--1; 25.1 degrees, pH 7.0 mu 0.5 M (phosphate)). Activation parameters based on k2 (deltaH not equal to = 1.8 kcal/mol, deltaS not equal to = --56 cal/mol-deg) indicate that the electron transfer process is substantially nondiabatic, in marked contrast with results obtained for Co(phen) 3 3+ as the oxidant. Linear kobsd VS. [Co(EDTA)--] plots are reported for the Co(EDTA)-- oxidation of cuprous stellacyanin at pH 10 (k = 8.9 M--1 sec--1; 25.0, pH 10, mu 0.5 M (carbonate); DELTaH not equal to 11.3 kcal/mol, deltaS not equal to = -16 cal/mol-deg) and at pH 7 in the presence of excess EDTA (k = 21.2 M--1 sec--1; 25.1 degree, pH 7.0, mu 0.5 M (phosphate), [EDTA] tot = 5 X 10(--4) M; deltaH not equal to = 5.9 kcal/mol, delta S not equal to = --33 cal/mol-deg). It is concluded that Co(EDTA)-- adopts an electron transfer mechanism similar to that preferred by Co(phen)33+ under conditions where the oxidant is prevented from binding strongly to reduced stellacyanin.     

Kinetic isotope effect and the presteady-state kinetics of the reaction catalyzed by the bacterial formate dehydrogenase

The primary kinetic isotope effect of the reaction catalyzed by NAD+-dependent formate dehydrogenase (EC 1.2.1.2.) from the methylotrophic bacterium Pseudomonas sp. 101 has been studied. Analysis of the ratios HVm/DVm and H(Vm/KM)/D(Vm/KM) in the pH range 6.1-7.9 showed that the transfer of hydride ion in ternary enzyme-substrate complex is a limiting step of the reaction, and the formate binding to the binary complex (formate dehydrogenase + NAD+) reached equilibrium when the pH of the medium was increased. An approach has been developed to determine the elementary constants of substrate association (kon) and dissociation (koff) at the stages of the binary--ternary enzyme-substrate complexes for the random equilibrium 2-substrate kinetic mechanism. The kon and koff values obtained for the bacterial formate dehydrogenase by using the proposed approach for NAD+ were (4.8 +/- 0.8)*10(5)M-1s-1 and (90 +/- 10) s-1, and for formate (2.0 +/- 1.0)*10(4) M-1s-1 and (60 +/- 20) s-1, respectively.     

Superoxide dismutase mimetic activity of cytokinin-copper(II) complexes

Dissociation constants of cytokinins, derivatives of purine which form complexes with cupric ion, were determined by spectrophotometry and the stability constants of their copper complexes by pH titration. The values found for kinetin were 3.76, 9.96, 7.8, and 15.3 for pK1, pK2, logk1, and log beta 2, respectively, and those for 6-benzylaminopurine were, in the same order, 3.90, 9.84, 8.3, and 15.9. The copper(II) complexes with kinetin and 6-benzylaminopurine had superoxide dismutase mimetic activity, and the reaction rate constants with superoxide, which were determined by polarography, were 2.3 X 10(-7) M-1 s-1 for kinetin and 1.5 X 10(-7) M-1 s-1 for 6-benzylaminopurine at pH 9.8 and 25 degrees C.     

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.     

Laser flash photolysis studies of electron transfer between ferredoxin-NADP+ reductase and several high-potential redox proteins

Complex formation and the kinetics of electron transfer between ferredoxin-NADP+ reductase (FNR) and two structurally homologous acidic 4Fe-4S high-potential ferredoxins (HiPIP's) from Ectothiorhodospira halophila (HP1 and HP2) and two structurally homologous cytochromes c2 from Paracoccus denitrificans and Rhodospirillum rubrum (PC2, and RC2, respectively) have been investigated by gel filtration and laser flash photolysis techniques. Gel filtration studies indicated that complex formation occurred between FNRox and HP1ox or HP2ox at low ionic strength (10 mM) and that the complexes were completely dissociated at high ionic strength (310 mM). Laser flash photolysis using lumiflavin as the reductant demonstrated that both free HP1ox and HP2ox reacted primarily with the anionic form of fully reduced lumiflavin (LFH-), whereas FNR was unreactive. Second-order rate constants of 1 X 10(6) and 0.8 X 10(6) M-1 s-1 were obtained for these reactions at 10 mM ionic strength. Increasing the ionic strength to 310 mM resulted in an approximately 1.5-fold increase in the rate constant. Inclusion of stoichiometric amounts of FNRox into the reaction mixture at low ionic strength led to a 2.5-fold increase in the rate constants. The reaction of 5-deazariboflavin semiquinone (5-dRf.) with the oxidized HiPIP's was also investigated by laser flash photolysis. Second-order rate constants of 3.0 X 10(8) M-1 s-1 (HP1) and 2.5 X 10(8) M-1 s-1 (HP2) were obtained for the free proteins at 10 mM ionic strength. Under the same conditions, 5-dRf. reacted with free FNRox, resulting in the formation of the neutral protein-bound semiquinone (FNR.), with a second-order rate constant of 6 X 10(8) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of 4-nitroquinoline 1-oxide with deoxyribodinucleotides.

The interactions of 4-nitroquinoline 1-oxide (NQO), a potent mutagen and carcinogen, with several self- and non-self-complementary deoxydinucleotides were probed by using absorption spectra of the charge transfer bands and 1H and 13C NMR spectra. Absorption spectra were analyzed by using Benesi-Hildebrand-type equations to yield stoichiometries and equilibrium constants of complex formation. Non-self complementary dimers form weak l:1 complexes [dpTpG:NQO, K(25 degrees C) = 22 M-1] while self-complementary dimers form strong 2:1 complexes [dpCpG)2:NQO, K(25 degrees C) = 2.2 X 10(4) M-2]. A mixture of dpTpG and dpCpA with NQO gives a 2:1 complexes [dpCpG)2:NQO, K(25 degrees C) = 2.2 X 10(4) M-2]. A mixture of dpTpG and dpCpA, K(25 degrees C) = 8.6 X 10(3) M-2]. Analyses of the changes in 13C and 1H NMR chemical shifts with complex formation gave approximate orientations for the intercalation of NQO with self-complementary dimer minihelixes. In the (dpCpG)2:NQO and (dpGpC)2:NQO complexes, the NO2 group of NQO probably lies in the major grove and the NO2, NO containing NQO ring is stacked near the purine imidazole ring. In the (dpTpA)2:NQO and (dpApT)2NQO complexes, the NO2 seems to project into the minor grove and the NQO benzenoid ring is over the purine imidazole ring.     

The energy landscape for ubihydroquinone oxidation at the Q(o) site of the bc(1) complex in Rhodobacter sphaeroides

Activation energies for partial reactions involved in oxidation of quinol by the bc(1) complex were independent of pH in the range 5. 5-8.9. Formation of enzyme-substrate complex required two substrates, ubihydroquinone binding from the lipid phase and the extrinsic domain of the iron-sulfur protein. The activation energy for ubihydroquinone oxidation was independent of the concentration of either substrate, showing that the activated step was in a reaction after formation of the enzyme-substrate complex. At all pH values, the partial reaction with the limiting rate and the highest activation energy was oxidation of bound ubihydroquinone. The pH dependence of the rate of ubihydroquinone oxidation reflected the pK on the oxidized iron-sulfur protein and requirement for the deprotonated form in formation of the enzyme-substrate complex. We discuss different mechanisms to explain the properties of the bifurcated reaction, and we preclude models in which the high activation barrier is in the second electron transfer or is caused by deprotonation of QH(2). Separation to products after the first electron transfer and movement of semiquinone formed in the Q(o) site would allow rapid electron transfer to heme b(L). This would also insulate the semiquinone from oxidation by the iron-sulfur protein, explaining the efficiency of bifurcation.     

Oxidation of 4-tert-butylcatechol and dopamine by hydrogen peroxide catalysed by horseradish peroxidase.

The catalytic cycle of horseradish peroxidase (HRP; donor:hydrogen peroxide oxidoreductase; EC 1.11.1.7) is initiated by a rapid oxidation of it by hydrogen peroxide to give an enzyme intermediate, compound I, which reverts to the resting state via two successive single electron transfer reactions from reducing substrate molecules, the first yielding a second enzyme intermediate, compound II. To investigate the mechanism of action of horseradish peroxidase on catechol substrates we have studied the oxidation of both 4-tert-butylcatechol and dopamine catalysed by this enzyme. The different polarity of the side chains of both o-diphenol substrates could help in the understanding of the nature of the rate-limiting step in the oxidation of these substrates by the enzyme. The procedure used is based on the experimental data to the corresponding steady-state equations and permitted evaluation of the more significant individual rate constants involved in the corresponding reaction mechanism. The values obtained for the rate constants for each of the two substrates allow us to conclude that the reaction of horseradish peroxidase compound II with o-diphenols can be visualised as a two-step mechanism in which the first step corresponds to the formation of an enzyme-substrate complex, and the second to the electron transfer from the substrate to the iron atom. The size and hydrophobicity of the substrates control their access to the hydrophobic binding site of horseradish peroxidase, but electron density in the hydroxyl group of C-4 is the most important feature for the electron transfer step.     

Kinetics of reduction of Clostridium pasteurianum rubredoxin by laser photoreduced spinach ferredoxin:NADP+ reductase and free flavins. Electron transfer within a protein-protein complex

The kinetics of reduction of oxidized Clostridium pasteurianum rubredoxin (Rdox) by free flavin semiquinones generated by the laser flash photolysis technique and by spinach ferredoxin:NADP+ reductase (FNR) semiquinone (also produced by flavin semiquinone reduction) have been investigated under anaerobic conditions. 5-Deazariboflavin semiquinone (5-dRf) rapidly reduces oxidized rubredoxin (Rdox) (k = 3.0 X 10(8) M-1 S-1) and oxidized ferredoxin:NADP+ reductase (FNRox) to the semiquinone level (k = 5.5 X 10(8) M-1 S-1). Lumiflavin semiquinone reduces Rdox more slowly (k = 1.3 X 10(7) M-1 S-1) and is not measurably reactive with FNRox. Absorption difference spectroscopy and difference CD indicate that Rdox and FNRox form a 1:1 complex at low ionic strength (10 mM), which is completely dissociated at higher ionic strength (310 mM). Apparent second order rate constants for reduction of Rdox in its free and complexed state by lumiflavin semiquinone are the same. Reduction of Rdox (both free and complexed) by free FNR semiquinone and intracomplex electron transfer were investigated using 5-dRf as the reductant. At I = 10 mM, a first order rate constant of 2.0 X 10(3) S-1 was obtained, which corresponds to the processes involved in intracomplex electron transfer from FNR semiquinone to Rdox. A second order reaction between free FNR semiquinone and complexed Rdox was also observed to occur (k = 5 X 10(7) M-1 S-1). At I = 310 mM, these reactions are not observed and the reaction of FNR semiquinone with free Rdox is second order (k = 4 X 10(6) M-1 S-1).     

Steady-state kinetics of thiocyanate oxidation catalyzed by human salivary peroxidase

A steady-state kinetic analysis was made of thiocyanate (SCN-) oxidation catalyzed by human peroxidase (SPO) isolated from parotid saliva. For comparative purposes, bovine lactoperoxidase (LPO) was also studied. Both enzymes followed the classical Theorell-Chance mechanism under the initial conditions [H2O2] less than 0.2mM, [SCN-] less than 10mM, and pH greater than 6.0. The pH-independent rate constants (k1) for the formation of compound I were estimated to be 8 X 10(6) M-1 s-1 (SD = 1, n = 18) for LPO and 5 X 10(6) M-1 s-1 (SD = 1, n = 11) for SPO. The pH-independent second-order rate constants (k4) for the oxidation of thiocyanate by compound I were estimated to be 5 X 10(6) M-1 s-1 (SD = 1, n = 18) for LPO and 9 X 10(6) M-1 s-1 (SD = 2, n = 11) for SPO. Both enzymes were inhibited by SCN- at pH less than 6. The pH-independent equilibrium constant (Ki) for the formation of the inhibited enzyme-SCN- complex was estimated to be 24 M-1 (SD = 12, n = 8) for LPO and 44 M-1 (SD = 4, n = 10) for SPO. An apparent pH dependence of the estimated values for k4 and Ki for both LPO and SPO was consistent with a mechanism based on assumptions that protonation of compound I was necessary for the SCN- peroxidation step, that a second protonation of compound I gave an inactive form, and that the inhibited enzyme-SCN- complex could be further protonated to give another inactive form.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the mechanism of quinol oxidation at the QP site in the cytochrome bc1 complex: studied using mutants lacking cytochrome bL or bH

To elucidate the mechanism of bifurcated oxidation of quinol in the cytochrome bc1 complex, Rhodobacter sphaeroides mutants, H198N and H111N, lacking heme bL and heme bH, respectively, were constructed and characterized. Purified mutant complexes have the same subunit composition as that of the wild-type complex, but have only 9-11% of the electron transfer activity, which is sensitive to stigmatellin or myxothiazol. The Em values for hemes bL and bH in the H111N and H198N complexes are -95 and -35 mV, respectively. The pseudo first-order reduction rate constants for hemes bL and bH in H111N and H198N, by ubiquiniol, are 16.3 and 12.4 s(-1), respectively. These indicate that the Qp site in the H111N mutant complex is similar to that in the wild-type complex. Pre-steady state reduction rates of heme c1 by these two mutant complexes decrease to a similar extent of their activity, suggesting that the decrease in electron transfer activity is due to impairment of movement of the head domain of reduced iron-sulfur protein, caused by disruption of electron transfer from heme bL to heme bH. Both mutant complexes produce as much superoxide as does antimycin A-treated wild-type complex. Ascorbate eliminates all superoxide generating activity in the intact or antimycin inhibited wild-type or mutant complexes.     

Kinetics of adrenodoxin reductase oxidation by non-physiologic electron acceptors

The reactions of NADPH oxidation by quinones and inorganic complexes catalyzed by NADPH: adrenodoxin reductase were studied. The catalytic constant for the enzyme at pH 7.0 is 20-25 s-1; the oxidative constants for the quinones vary from 5 X 10(5) to 1.1 X 10(3) M-1 s-1 and show an increase with a rise in the one-electron acceptor reduction potential. The mode of adrenodoxin reductase interaction with oxyquinones differs from that of the enzyme interaction with alkyl-substituted quinones and inorganic complexes. NADPH competitively inhibits electron acceptors, whereas NADP+ is a competitive inhibitor of NADPH and a uncompetitive inhibitor of electron acceptors. (Ki = 25 microM). The depth of FAD incorporation into the enzyme molecule as calculated according to the outer sphere electron transfer theory is 6.1 A.     

Characterization of the oxycomplex of lignin peroxidases from Phanerochaete chrysosporium: equilibrium and kinetics studies

The oxycomplexes (compound III, oxyperoxidase) of two lignin peroxidase isozymes, H1 (pI = 4.7) and H8 (pI = 3.5), were characterized in the present study. After generation of the ferroperoxidase by photochemical reduction with deazoflavin in the presence of EDTA, the oxycomplex is formed by mixing ferroperoxidase with O2. The oxycomplex of isozyme H8 is very stable, with an autoxidation rate at 25 degrees C too slow to measure at pH 3.5 or 7.0. In contrast, the oxycomplex of isozyme H1 has a half-life of 52 min at pH 4.5 and 29 min at pH 7.5 at 25 degrees C. The decay of isozyme H1 oxycomplex follows a single exponential. The half-lives of lignin peroxidase oxycomplexes are much longer than those observed with other peroxidases. The binding of O2 to ferroperoxidase to form the oxycomplex was studied by stopped-flow methods. At 20 degrees C, the second-order rate constants for O2 binding are 2.3 X 10(5) and 8.9 X 10(5) M-1 s-1 for isozyme H1 and 6.2 X 10(4) and 3.5 X 10(5) M-1 s-1 for isozyme H8 at pH 3.6 and pH 6.8, respectively. The dissociation rate constants for the oxycomplex of isozyme H1 (3.8 Z 10(-3) s-1) and isozyme H8 (1.0 X 10(-3) s-1) were measured at pH 3.6 by CO trapping. Thus, the equilibrium constants (K, calculated from kon/koff) for both isozymes H1 (7.0 X 10(7) M-1) and H8 (6.2 X 10(7) M-1) are higher than that of myoglobin (1.9 Z 10(6) M-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of electron transfer between cytochrome c and laccase.

Rate constants have been determined for the electron-transfer reactions between reduced horse heart cytochrome c and resting Rhus vernicifera laccase as a function of pH, ionic strength, and temperature. The second-order rate constant for the oxidation of reduced cytochrome c was determined to be k = 125 M-1 s-1 at 25 degrees C in 0.2 M phosphate buffer at pH 6.0 with the activation parameters delta H++ = 16.2 kJ mol-1 and delta S++ = 28.9 J mol-1 K-1. The rate constants increased with decreasing buffer concentration, indicating that electron transfer from cytochrome c to laccase is favored by the local electrostatic interaction (ZAZB = -0.9 at pH 6 and -1.3 at pH 4.8) between the basic proteins with positive net charges. From the increase of the rate of electron transfer with decreasing pH, one of the driving forces of the reaction was suggested to be the difference in the redox potentials between the type 1 copper in laccase and the central iron in cytochrome c. Further, on addition of one hexametaphosphate anion per cytochrome c molecule, the rate of the electron transfer was increased, probably because the association of both proteins became more favorable.