The location of the polyphosphate-binding sites on cytochrome c measured by NMR paramagnetic difference spectroscopy.
Analyses of unimolecular electron self-exchange reactions provide a comparatively simple and direct approach to understanding biological electron transfer. Such studies are currently limited by a lack of well characterised aggregating systems. In the presence of sodium hexametaphosphate, cytochrome c forms stable protein aggregates as a result of binding hexametaphosphate at a single site on its surface (preceding paper in this issue of the journal). Here we report the location of the principal polyphosphate binding site on the surface of cytochrome c for both hexametaphosphate and a second polyphosphate, tripolyphosphate determined using 1H-NMR spectroscopy in conjunction with the relaxation probe potassium hexacyanochromium(III). Addition of either hexametaphosphate or tripolyphosphate to ferricytochrome c in the presence of the relaxation probe causes a decrease in intensity of several resonances in the paramagnetic difference spectrum, including Phe82 ortho/meta, Ile85 delta methyl and Ile9 gamma methyl. Together these effects put the site of polyphosphate binding close to lysines 13, 86, and 87. Additionally the effect of sodium tripolyphosphate and sodium trimetaphosphate on cytochrome c aggregation is described. The potential role of this site in anion-induced cytochrome c aggregation is discussed.
Concar
D W
DW
Inorganic Chemistry Laboratory, University of Oxford, England.
Whitford
D
D
Williams
R J
RJ
D000818:Animals; D001665:Binding Sites; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D006736:Horses; D006859:Hydrogen; D007700:Kinetics; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008968:Molecular Conformation; D009206:Myocardium / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism* / Q000494:pharmacology; D011122:Polyphosphates / Q000378:metabolism* / Q000494:pharmacology; D011487:Protein Conformation
D003574:Cytochrome c Group; D046911:Macromolecular Substances; D010710:Phosphates; D011122:Polyphosphates; C005035:hexametaphosphate; D006859:Hydrogen; C005692:triphosphoric acid