Rapid mixing of ferrocytochrome c peroxi- sition (15). Corroboration of this rate by a more direct measurement is desirable in the context of understanding biological electron transfer rates.We, therefore, wish to report the direct measurement of electron transfer from cyt c peroxidase(II) to cyt c(III) within the cyt c(III)-cyt c peroxidase(II) complex. In addition, we report data for electron transfer in the derivative system, cyt c peroxidase-porphyrin (Por) cyt c7 (AP0 1 V), where Por-cyt c7 is the anion radical of Por-cyt c. Por-cyt c is iron-free cyt c. These systems together provide unique data on the dependence of protein electron transfer rates on AG.Finally, Ho et al. (16) reported an interesting study on the rate of oxidative quenching of the Zn-cyt c peroxidase triplet by cyt c(III). They found that 3kq depends markedly on the primary structure of the cytochrome with 3kq (yeast C) > 10 3kq (horse C). With the methods in hand to study a simple Fe(II)/Fe(III) redox reaction in the cyt c/cyt c peroxidase system, we report a comparative study of how the cyt c peroxidase(II) + cyt c(III) reaction depends on the primary structure of cyt c. (18). The product was purified by G-200 Abbreviations: cyt c, cytochrome c; Por, porphyrin. 1To whom reprint requests should be addressed. MATERIALS AND METHODS 1330The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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.

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Abstract:The kinetics of reduction of femcyanide by yeast ferrocytochrome c peroxidase (CPPI') were investigated as a function of ionic strength in phosphate buffers at pH 7.0 and 25 3= 1°C. The observed bimolecular rate constant (k12) is 8.4 x 10" M-I s-I in 0.1 M phosphate. The dependence of the reaction rate on ionic strength indicates a change of -9 on the protein at pH 7.0, which is in good agreement with the total charge of -1 1 estimated for CCP" from its amino acid content. Substituting k12 at infinite ionic strength (kz) into the Marcus cross relation yields an electron self-exchange rate constant ( k: ) for the Felll/Fell couple of CCP of 7.2 x M-' s-I. This value is over four orders of magnitude higher than that calculated for the ~e'~/Fe'll couple of CCP from literature data for cross-reactions with ferrocyanide at pH 7.0. Possible reasons for the large difference in the two CCP k;"; values are discussed. Literature data also allowed k;"; values for various other heme proteins to be determined from their cross-reactions with femcyanide. The calculated rate constants vary by eight orders of magnitude, and the variation of k; with protein structure suggests that the redox reactivity of ferrous heme proteins towards femcyanide is dependent on the spin state and coordination of iron, as well as on the accessibility of the heme.