HORSE HEART FERRICYTOCHROME-C - CONFORMATION AND HEME CONFIGURATION OF HIGH IONIC-STRENGTH ACIDIC FORMS

The absorption, circular dichroism, and resonance Raman spectra of horse heart ferricytochrome c in the presence of 0.2 M KCl, 0.1 M NaClO4, and 0.2 M KNO3, in the pH region 7 to 0.5, have been investigated to determine the nature and the course of the processes involved. As in the absence of salts (Myer, Y., and Saturno, A. F. (1990) J. Protein Chem, 9, 379-387), the change from neutral to low acidic pH's in the presence of salts is a three-step process: state III(s) <--> state III(s,a) <--> state II(s), <--> state I(s), with pK(a)'s of 3.5 +/- 0.2, 2.2 +/- 0.2, and 1.1 +/- 0.2, and with two, one, and one number of protons, respectively. The addition of salts at neutral pH's has little or no effect on the protein conformation and the heme-iron configuration (i.e., they remain the same, low-spin hexacoordinated heme iron with a Met-80-Fe-His-18 axial coordination), but such addition does cause a slight tightening of the heme crevice and the enlargement of the porphyrin core. State III(s,a) is a folded state with about the same degree of folding and with a similar spin state and coordination configuration of iron, but the heme crevice is loosened and the porphyrin core is smaller. Both states II(s) and I(s) are also essentially folded forms, but with a smaller degree of protein secondary structure. State II(s) has a high-spin hexacoordinated heme iron with a water molecule and a protonated and/or hydrogen-bonded imidazole of his-18 as the two axial ligates; and state I(s) has a high-spin pentacoordinated heme iron, which is about 0.49 angstrom out of the porphyrin plane, with a protonated and/or hydrogen-bonded imidazole nitrogen as the only axial ligate. The addition of anions causes the stabilization of the protein secondary structures and the state III(a) --> state II transition. The mode of effectiveness of anions appears to be nonspecific (i.e., because of electrostatic shielding and/or disruption of salt bridges).