Characterization of four covalently-linked yeast cytochrome c/cytochrome c peroxidase complexes: Evidence for electrostatic interaction between bound cytochrome c molecules.
Four covalent complexes between recombinant yeast cytochrome c and cytochrome c peroxidase (rCcP) were synthesized via disulfide bond formation using specifically designed protein mutants (Papa, H. S., and Poulos, T. L. (1995) Biochemistry 34, 6573-6580). One of the complexes, designated V5C/K79C, has cysteine residues replacing valine-5 in rCcP and lysine-79 in cytochrome c with disulfide bond formation between these residues linking the two proteins. The V5C/K79C complex has the covalently bound cytochrome c located on the back-side of cytochrome c peroxidase, approximately 180 degrees from the primary cytochrome c-binding site as defined by the crystallographic structure of the 1:1 noncovalent complex (Pelletier, H., and Kraut J. (1992) Science 258, 1748-1755). Three other complexes have the covalently bound cytochrome c located approximately 90 degrees from the primary binding site and are designated K12C/K79C, N78C/K79C, and K264C/K79C, respectively. Steady-state kinetic studies were used to investigate the catalytic properties of the covalent complexes at both 10 and 100 mM ionic strength at pH 7.5. All four covalent complexes have catalytic activities similar to those of rCcP (within a factor of 2). A comprehensive study of the ionic strength dependence of the steady-state kinetic properties of the V5C/K79C complex provides evidence for significant electrostatic repulsion between the two cytochromes bound in the 2:1 complex at low ionic strength and shows that the electrostatic repulsion decreases as the ionic strength of the buffer increases.
Nakani
Siddhartha
S
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, USA.
Vitello
Lidia B
LB
Erman
James E
JE
D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D045304:Cytochromes c; D003578:Cytochrome-c Peroxidase