Ion binding to cytochrome c studied by nuclear magnetic quadrupole relaxation

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Abstract
The enhancement of the 35Co- transverse relaxation rate on binding of Cl- to oxidized and reduced cytochrome c was studied under conditions of variable NaCl concentration, temperature, pH, sodium phosphate, iron hexacyanide and sodium cyanide concentration. The presence of a strong binding site(s) for Cl- in oxidized and reduced cytochrome c, with a higher affinity in ferrocytochrome c was revealed. Competition experiments suggest that these sites also bind iron hexacyanide and phosphate. Cyanide binding to the Fe in ferricytochrome c at alkaline and neutral pH decreased the binding of Cl-. The pH dependence of the 35Cl- relaxation rate was fitted by using literature pK values for ionizable groups. No indications of Na+ binding to oxidized and reduced cytochrome c were observed by using 23Na+ NMR. Cl- is apparently bound near the exposed heme edge and the surface structure or dynamics in this region are different in the 2 oxidation states.
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