To understand the determinants of redox potential and protein stability in c-type cytochromes, we have characterized two mutations to a highly conserved tyrosine group, tyrosine-75, of Rhodobacter capsulatus cytochrome c2. Mutant Y75F was designed to test the importance of the tyrosine hydroxyl group to the typically high redox potentials of the cytochromes c2 while maintaining a hydrophobic core. Mutant Y75C was designed to test the importance of a large hydrophobic group to redox potential by replacing an aromatic group with a small nonpolar group. Both mutants exhibit spectral and redox properties indicating that their heme environments have been perturbed. The kinetics of reduction by lumiflavin semiquinone and photooxidation by Rhodobacter sphaeroides photosynthetic reaction centers have been used to demonstrate that both mutants are structurally analogous to the wild-type protein at the active site of electron transfer. Different degrees of relative stability of the mutants toward a denaturant have been observed with the order being Y75C less than wt less than Y75F in the oxidized state and Y75C less than Y75F less than wt in the reduced state. These results are discussed in light of the recent structure determination of the R. capsulatus wild-type ferrocytochrome c2 to suggest that R. capsulatus tyrosine-75, or its equivalent in other species, is part of a conserved hydrogen-bonding network which plays an important role in maintaining high redox potentials and protein stability of cytochromes c in general. Study holds ProTherm entries: 4387, 4388, 4389, 4390, 4391, 4392 Extra Details: conserved tyrosine; redox potentials; hydrophobic core;,heme environments; active site; hydrogen-bonding network
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:25 p.m.
|Number of data points||22|
|Proteins||Cytochrome c2 ; Cytochrome c2|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence GDAAKGEKEFNKCKTCHSIIAPDGTEIVKGAKTGPNLYGVVGRTAGTYPEFKYKDSIVALGASGFAWTEEDIATYVKDPGAFLKEKLDDKKAKTGMAFKLAKGGEDVAAYLASVVK|