Role of the Low-Affinity Binding Site in Electron Transfer from Cytochrome c to Cytochrome c Peroxidase

Abstract

The interaction of yeast iso-1-cytochrome c (yCc) with the high- and low-affinity binding sites on cytochrome c peroxidase compound I (CMPI) was studied by stopped-flow spectroscopy. When 3 microM reduced yCc(II) was mixed with 0.5 microM CMPI at 10 mM ionic strength, the Trp-191 radical cation was reduced from the high-affinity site with an apparent rate constant>3000 s(-1), followed by slow reduction of the oxyferryl heme with a rate constant of only 10 s(-1). In contrast, mixing 3 microM reduced yCc(II) with 0.5 microM preformed CMPI *yCc(III) complex led to reduction of the radical cation with a rate constant of 10 s(-1), followed by reduction of the oxyferryl heme in compound II with the same rate constant. The rate constants for reduction of the radical cation and the oxyferryl heme both increased with increasing concentrations of yCc(II) and remained equal to each other. These results are consistent with a mechanism in which both the Trp-191 radical cation and the oxyferryl heme are reduced by yCc(II) in the high-affinity binding site, and the reaction is rate-limited by product dissociation of yCc(III) from the high-affinity site with apparent rate constant k(d). Binding yCc(II) to the low-affinity site is proposed to increase the rate constant for dissociation of yCc(III) from the high-affinity site in a substrate-assisted product dissociation mechanism. The value of k(d) is <5 s(-1) for the 1:1 complex and >2000 s(-1) for the 2:1 complex at 10 mM ionic strength. The reaction of horse Cc(II) with CMPI was greatly inhibited by binding 1 equiv of yCc(III) to the high-affinity site, providing evidence that reduction of the oxyferryl heme involves electron transfer from the high-affinity binding site rather than the low-affinity site. The effects of CcP surface mutations on the dissociation rate constant indicate that the high-affinity binding site used for the reaction in solution is the same as the one identified in the yCc*CcP crystal structure.

Control of Cyclic Photoinitiated Electron Transfer between Cytochrome c Peroxidase (W191F) and Cytochrome c by Formation of Dynamic Binary and Ternary Complexes

“…Kinetic measurements and mutagenesis, both in our laboratory 8, 43–45 and others’ 12, 46–48 generally arrived at measured affinity constants for the ‘first’ and ‘second’ Cc, which correspond to thermodynamic constants of K I ~ 10 7 M −1 , K II ~ 10 4 M −1 at 10mM potassium phosphate buffer, pH 7 20C, values corroborated in the present study. Use of these consensus values permits us to discuss the actual site constants and to set limits on the repulsion free energy.…”

Design of a Ruthenium-Labeled Cytochrome c Derivative to Study Electron Transfer with the Cytochrome bc1 Complex

“…However, the electrostatic interactions of the two cytochromes c are similar, as indicated 2) by the similar slopes in the rate constant versus ionic strength plots (Figure 7). This is comparable to the situation with yeast cytochrome c peroxidase, where yeast Cc has a much stronger hydrophobic interaction than horse Cc (48). The origin of this difference between yeast and horse Cc is unknown.…”

“…These complexes include the R. sphaeroides cyt c 2 :reaction center complex (50), the Cc:cyt c peroxidase complex (35,47,48,51), and the Cc:cyt c oxidase complex (52) in addition to the Cc:cyt bc 1 complex (17). The peripheral electrostatic interactions may guide the docking of Cc to the specific configuration stabilized by the central nonpolar domain that is optimized for rapid electron transfer.…”

Cytochrome c signalosome in mitochondria

“…The indolyl radical cation on Trp191 is finally reduced by a second ferrous Cc molecule (Wang et al 1996b), which transfers another electron to oxy-ferryl heme, returning CcP to its original state (Mei et al 2002). Whether the latter Cc molecule binds to a low-affinity second site on CcP (Leesch et al 2000;Mei et al 2002) or both Cc molecules sequentially interact with the high-affinity site of CcP is still debated. However, recent studies almost exclude the ET function for the low-affinity site, at least at physiological ionic strength (Pearl et al 2007;.…”