Amide proton exchange rates of oxidized and reduced Saccharomyces cerevisiae iso-1-cytochrome c.
Proton NMR spectroscopy was used to determine the rate constant, kobs, for exchange of labile protons in both oxidized (Fe(III)) and reduced (Fe(II)) iso-1-cytochrome c. We find that slowly exchanging backbone amide protons tend to lack solvent-accessible surface area, possess backbone hydrogen bonds, and are present in regions of regular secondary structure as well as in omega-loops. Furthermore, there is no correlation between kobs and the distance from a backbone amide nitrogen to the nearest solvent-accessible atom. These observations are consistent with the local unfolding model. Comparisons of the free energy change for denaturation, delta Gd, at 298 K to the free energy change for local unfolding, delta Gop, at 298 K for the oxidized protein suggest that certain conformations possessing higher free energy than the denatured state are detected at equilibrium. Reduction of the protein results in a general increase in delta Gop. Comparisons of delta Gd to delta Gop for the reduced protein show that the most open states of the reduced protein possess more structure than its chemically denatured form. This persistent structure in high-energy conformations of the reduced form appears to involve the axially coordinated heme.
Marmorino
J L
JL
Department of Chemistry, University of North Carolina, Chapel Hill 27599.
Auld
D S
DS
Betz
S F
SF
Doyle
D F
DF
Young
G B
GB
Pielak
G J
GJ
D000577:Amides / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry*; D045304:Cytochromes c; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013499:Surface Properties; D013816:Thermodynamics
D000577:Amides; C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D005290:Ferric Compounds; D005296:Ferrous Compounds; D011522:Protons; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c