Oxidation-state-dependent protein docking between cytochrome c and cytochrome b₅: High-pressure laser flash photolysis study

To characterize the protein-protein interaction during electron transfer, we used Zn-substituted cytochrome c (ZnCytc) as a model of ferrous Cytc and determined the volume change, ΔV_d^Zn, for the dissociation of its complex with ferric cytochrome b₅ (Cytb₅) by the pressure dependence of its photoinduced electron-transfer kinetics. Under ambient pressure, the dissociation constant, K_d^Zn, of the ZnCytc/Cytb₅ complex was dependent on the buffer concentration, 1.5 and 12 μM in 2 and 10 mM Tris-HCl, pH 7.4, respectively, which was consistent with formation of salt bridges in its complexation. The dissociation of one salt bridge is usually associated with large volume changes of -10 to -30 cm³ mol^-1, while pressure dependence of K_d^Zn resulted in smaller value of ΔV_d^Zn, -8.5 cm³ mol^-1. Therefore, the interaction between ZnCytc and Cytb₅ cannot be explained only by salt bridge interaction, and the partial cancellation by the positive volume change due to the additional hydrophobic interaction is a plausible explanation for the observed ΔV_d^Zn. In addition, ΔV_d^Zn of -8.5 cm³ mol^-1 was considerably smaller than the previously reported volume change, ΔV_d^Fe, of -122 cm³ mol^-1 in the ferric Cytc/Cytb₅ complex dissociation [Rodgers and Sligar (1991) J. Mol. Biol. 221, 1453-1460]. ZnCytc used here has been assumed to be a reliable model of ferrous Cytc, and thus the discrepancy between our present ΔV_d^Zn and the previous ΔV_d^Fe is discussed on the basis of the protein docking dependent on the oxidation states of heme iron in Cytc.