Probing the Dynamics of a His73–Heme Alkaline Transition in a Destabilized Variant of Yeast Iso-1-cytochrome c with Conformationally Gated Electron Transfer Methods
journal contributionposted on 22.11.2011, 00:00 by Swati Bandi, Bruce E. Bowler
The alkaline transition of cytochrome c involves substitution of the Met80 heme ligand of the native state with a lysine ligand from a surface Ω-loop (residues 70 to 85). The standard mechanism for the alkaline transition involves a rapid deprotonation equilibrium followed by the conformational change. However, recent work implicates multiple ionization equilibria and stable intermediates. In previous work, we showed that the kinetics of formation of a His73–heme alkaline conformer of yeast iso-1-cytochrome c requires ionization of the histidine ligand (pKHL ∼ 6.5). Furthermore, the forward and backward rate constants, kf and kb, respectively, for the conformational change are modulated by two auxiliary ionizations (pKH1 ∼ 5.5, and pKH2 ∼ 9). A possible candidate for pKH1 is His26, which has a strongly shifted pKa in native cytochrome c. Here, we use the AcH73 iso-1-cytochrome c variant, which contains an H26N mutation, to test this hypothesis. pH jump experiments on the AcH73 variant show no change in kobs for the His73–heme alkaline transition from pH 5 to 8, suggesting that pKH1 has disappeared. However, direct measurement of kf and kb using conformationally gated electron transfer methods shows that the pH independence of kobs results from coincidental compensation between the decrease in kb due to pKH1 and the increase in kf due to pKHL. Thus, His26 is not the source of pKH1. The data also show that the H26N mutation enhances the dynamics of this conformational transition from pH 5 to 10, likely as a result of destabilization of the protein.