Kinetics of reduction by free flavin semiquinones of the components of the cytochrome c-cytochrome c peroxidase complex and intracomplex electron transfer

Abstract

The kinetics of reduction by free flavin semiquinones of the individual components of 1:1 complexes of yeast ferric and ferryl cytochrome c peroxidase and the cytochromes c of horse, tuna, and yeast (iso-2) have been studied. Complex formation decreases the rate constant for reduction of ferric peroxidase by 44%. On the basis of a computer model of the complex structure [Poulos, T.L., & Finzel, B.C. (1984) Pept. Protein Rev. 4, 115-171], this decrease cannot be accounted for by steric effects and suggests a decrease in the dynamic motions of the peroxidase at the peroxide access channel caused by complexation. The orientations of the three cytochromes within the complex are not equivalent. This is shown by differential decreases in the rate constants for reduction by neutral flavin semiquinones upon complexation, which are in the order tuna much greater than horse greater than yeast iso-2. Further support for differences in orientation is provided by the observation that, with the negatively charged reductant FMNH., the electrostatic environments near the horse and tuna cytochrome c electron-transfer sites within their respective complexes with peroxidase are of opposite sign. For the horse and tuna cytochrome c complexes, we have also observed nonlinear concentration dependencies of the reduction rate constants with FMNH.. This is interpreted in terms of dynamic motion at the protein-protein interface. We have directly measured the physiologically significant intra-complex one electron transfer rate constants from the three ferrous cytochromes c to the peroxide-oxidized species of the peroxidase. At low ionic strength these rate constants are 920, 730, and 150 s-1 for tuna, horse, and yeast cytochromes c, respectively. These results are also consistent with the contention that the orientations of the three cytochromes within the complex with CcP are not the same. The effect on the intracomplex electron-transfer rate constant of the peroxidase amino acid side chain(s) that is (are) oxidized by the reduction of peroxide was determined to be relatively small. Thus, the rate constant for reduction by horse cytochrome c of the peroxidase species in which only the heme iron atom is oxidized was decreased by only 38%, indicating that this oxidized side-chain group is not tightly coupled to the ferryl peroxidase heme iron. Finally, it was found that, in the absence of cytochrome c, neither of the ferryl peroxidase species could be rapidly reduced by flavin semiquinones.(ABSTRACT TRUNCATED AT 400 WORDS)

Proton‐NMR studies of the effects of ionic strength and pH on the hyperfine‐shifted resonances and phenylalanine‐82 environment of three species of mitochondrial ferricytochrome c

“…We have found that, in each case, on NOE connectivity to phenyl ring protons of Phe82 near 6.0ppm is observed upon irradiation of the heme 3-CH3 resonance (Satterlee and Moench, 1987;Moench and Satterlee, 1989). In contrast, two-dimensional (2 D) NOESY experiments are less useful in this respect (Busseet al, 1990).…”

“…pH values are reported as 'p2H' which is the pH meter reading uncorrected for the deuterium effect. Singlepulse and pre-steady-state one-dimensional (1 D) NOE measurements were carried out as previously described (Satterlee and Moench, 1987). Irradiation pulse durations for data acquistions were generally 100 ms and decoupler power levels used for irradiation were 200 mW or less.…”

“…Despite this structural similarity, however, cytochrome c redox partner proteins are often capable of discriminating between cytochromes c from a wide variety of species. For example, cytochrome c peroxidase from baker's yeast is most active with its physiological redox partner, yeast cytochrome c, and peak enzymatic activity (Kang et al, 1977;Hazzard et al, 1987) and electron transfer rates (Hazzard et al, 1988b) are achieved at relatively high ionic strengths. This is in contrast to results obtained with cytochrome c peroxidase in combination with either tuna or horse cytochromes c which demonstrate that, in addition to a lower maximal turnover number relative to the complex between cytochrome c peroxidase and yeast cytochrome c, these complexes are maximally active at relatively low ionic strengths (Kang et al, 1977;Hazzard et al, 1987Hazzard et al, ,1988aKang and Erman, 1982).…”

“…1979;Hazzard et al, 1987Hazzard et al, , 1988aHazzard et al, , 1988b. For example, it has been suggested that small rearrangements in the positions of amino acid side chains, particularly aromatic side chains, near the heme 71 system could function to 'fine-tune' electron transfer mechanisms and rates (Poulos and Kraut, 1980;Poulos and Finzel, 1984).…”

“…First, by monitoring changes in these resonances with changes in general solution conditions, we have attempted to gain insight into the structural and/or electronic changes which contribute to specific environmentally induced changes in the isotropic shift pattern. This type of study is necessary in order to lay a groundwork on which to interpret resonance shift changes which occur when cytochrome c is titrated with redox partner proteins (Satterlee et al, 1987;Gupta and Yonetani, 1973;Eley and Moore, 1983;King et al, 1985;Das et al, 1988;Hazzard and Tollin, 1985). If structural implications of complex-induced NMR resonance shifts are ever to be interpreted, such as for redox complex formation between cytochrome c peroxidase and cytochrome c, general environmental influences, such as those addressed here must be determined so that they can be taken into consideration.…”