Temperature dependence of the unimolecular reduction of ferricytochrome c by ferrocytochrome c peroxidase at low and high ionic strengths

Abstract

The oxidation of ferrocytochrome c peroxidase by horse ferricytochrome c has been investigated at 0.02 and 0.2 M ionic strengths ( p ) in phosphate buffers, pH 7.0, over a range of temperatures from 0 to 26 OC. The absorbance-time curves for the reduction of cytochrome c a r e exponential at both 1's. At 3-10 pM peroxidase and 24 OC, the observed rate constants are 0.23 i 0.02 s-l ( p = 0.02 M) and 0.020 i 0.002 s-l ( p = 0.2 M), and the kinetics are independent of the cytochrome c concentration between 0.5 and I O pM. The activation enthalpies and entropies are 4.6 kcal mol-' and -46 eu at p = 0.02 M and 17 kcal mol-' and -7.8 eu at p = 0.2 M, which indicates that the rate-limiting steps are different at low and high p. The increased polarization of porphyrin cytochrome c fluorescence at p = 0.02 M in the presence of the peroxidase clearly indicates that the proteins form a stable complex at low p; at p = 0.2 M, no polarization increase is observed. Therefore, the unimolecular rate-limiting step at high p must occur prior to protein association. Oxidation of the peroxidase by both Fe(CN)63-and Fe(EDTA)-shows bimolecular kinetics at p = 0.2 M with rate constants of (8.4 i 0.4) X lo4 and 70 i 8 M-' s-l, respectively. Furthermore, these oxidations do not show limiting kinetics at reagent concentrations that yield half-lives IO-fold smaller than those for the cytochrome oxidation. Therefore, we speculate that at high p the smaller reagents have better access to the peroxidase heme than cytochrome c and that the peroxidase must undergo a conformation change which increases its heme exposure (such as its acidicalkaline transition) before it is oxidized by cytochrome c. J. Am. Chem. SOC. 1986, 108, 4665. (b) Ho, P. S.; Sutoris, C.; Liang, N.; Margoliash, E.; Hoffman, B. M. J . Am. Chem. SOC. 1985, 107, 1070. (c) Peterson-Kennedy, S. E.; McGourty, J. L.; Ho, P. S.; Sutoris, C. J.; Liang, N.; Zemel, H.; Blough, N. V.; Margoliash, E.; Hoffman, B. M. Coord. Chem. Rev. 1985, 64, 125. (a) Poulos. T. L.; Freer, S. T.; Alden, R. A,; Edwards, S. L.; Skoglund, U.; Takio, K.; Erikson, B. Xuong, N. H.; Yonetani, T.; Kraut, J. J. B i d . Chem. 1980, 255, 575. (b) Finzel, B. C.; Poulos, T. L.; Kraut, J. J. B i d . Chem. 1984, 259, 13027. Swanson, R.; Trus, B. L.; Mandel, N.; Mandel, G . ; Kallai, 0. B.; Dickerson, R. E.

Electrostatic and steric control of electron self-exchange in cytochromes c, c551, and b5

“…The reaction of cytochrome c with cytochrome c peroxidase has also been the focus of extensive study (Kang et al, 1977;Poulos and Kraut, 1980;Vitello and Erman, 1987;Liang et al, 1987;Moench et al, 1987;Poulos et al, 1987;Cheung and English, 1988;Hazzard et al, 1988). Using van Leeuwen's formalism, the data given by Kang et al 1977was not fit well using the charges and dipole moments given by Northrup et al (1986a); this is presumably because cytochrome c peroxidase reacts with cytochrome c at two different sites, as found in the Brownian dynamic simulations of Northrup et al (1986b, 1987, 1988).…”

Reductions by ferrocytochrome c peroxidase: 5. Kinetics of ferricyanide reduction

“…horse and yeast ferricytochromes c by CCP", which followed first-order kinetics in low and high Pi buffers (16,22). At low Pi, cytochrome c and CCP form a stable complex so intracomplex electron transfer was expected (16).…”

“…horse and yeast ferricytochromes c by CCP", which followed first-order kinetics in low and high Pi buffers (16,22). At low Pi, cytochrome c and CCP form a stable complex so intracomplex electron transfer was expected (16). However, the electrostatically stabilized cytochrome c -CCP complex dissociates at high salt, so a conformational change in CCP" was proposed to explain the first-order kinetics observed for the reduction of femcytochrome c at high salt (16,22).…”

“…At low Pi, cytochrome c and CCP form a stable complex so intracomplex electron transfer was expected (16). However, the electrostatically stabilized cytochrome c -CCP complex dissociates at high salt, so a conformational change in CCP" was proposed to explain the first-order kinetics observed for the reduction of femcytochrome c at high salt (16,22). Since only bimolecular kinetics were observed for the reduction of ferricyanide, the mechanism of CCP" reduction of the large (ferricytochrome c) and small (ferricyanide) oxidants is clearly different.…”

Cross-linked metalloproteins: novel systems for the study of intraprotein electron-transfer reactions

“…The driving force and the intervening medium can be varied purposefully by metal substitution and by site-specific mutagenesis. Although the investigation of intracomplex redox reactions b%an only several years ago, interesting findings have been made, and important new questions have been raised (80)(81)(82)(83)(84)(85)(86)(87)(88)(89)(90)(91). The kinetics of these reactions is one aspect of the present study.…”