Probing electrostatic interactions in cytochrome c using site-directed chemical modification.
This communication reports the generation of an electrostatic probe using chemical modification of methionine side chains. The alkylation of methionine by iodoacetamide was achieved in a set of Saccharomyces cerevisiae iso-1-cytochrome c mutants, introducing the nontitratable, nondelocalized positive charge of a carboxyamidomethylmethionine sulfonium (CAMMS) ion at five surface and one buried site in the protein. Changes in redox potential and its variation with temperature were used to calculate microscopic effective dielectric constants operating between the probe and the heme iron. Dielectric constants (epsilon) derived from deltadeltaG values were not useful due to entropic effects, but epsilon(deltadeltaH) gave results that supported the theory. The effect on biological activity of surface derivatization was interpreted in terms of protein-protein interactions. The introduction of an electrostatic probe in cytochrome c often resulted in marked effects on activity with one of two physiological partners: cytochrome c reductase, especially if introduced at position 65, and cytochrome c oxidase, if at position 28.
Blouin
Christian
C
Department of Biochemistry, Dalhousie University, Halifax, NS, Canada.
Guillemette
J Guy
JG
Wallace
Carmichael J A
CJ
D000478:Alkylation; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008715:Methionine / Q000031:analogs & derivatives / Q000737:chemistry*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D013452:Sulfonium Compounds / Q000737:chemistry*; D013816:Thermodynamics
D003574:Cytochrome c Group; D013452:Sulfonium Compounds; D006418:Heme; D008715:Methionine; D010088:Oxidoreductases; C022691:succinate oxidase; D003576:Electron Transport Complex IV