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.
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 (pK(HL) ~ 6.5). Furthermore, the forward and backward rate constants, k(f) and k(b), respectively, for the conformational change are modulated by two auxiliary ionizations (pK(H1) ~ 5.5, and pK(H2) ~ 9). A possible candidate for pK(H1) is His26, which has a strongly shifted pK(a) 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 k(obs) for the His73-heme alkaline transition from pH 5 to 8, suggesting that pK(H1) has disappeared. However, direct measurement of k(f) and k(b) using conformationally gated electron transfer methods shows that the pH independence of k(obs) results from coincidental compensation between the decrease in k(b) due to pK(H1) and the increase in k(f) due to pK(HL). Thus, His26 is not the source of pK(H1). 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.
Department of Chemistry and Biochemistry and Center for Biomolecular Structure and Dynamics, The University of Montana, Missoula, Montana 59812, USA.
Swati
S
Bandi
Bruce E
BE
Bowler
D003575:Cytochromes c1 / Q000737:chemistry* / Q000235:genetics*; D004579:Electron Transport; D006418:Heme / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics*; D013816:Thermodynamics
D029701:Saccharomyces cerevisiae Proteins; D006418:Heme; D006639:Histidine; D003575:Cytochromes c1