Analysis of field-dependent relaxation data and hyperfine shifts of cytochrome c' from Rhodospirillum rubrum in terms of the high-spin iron ligation states

Abstract

The pH and field dependence of the paramagnetic line widths of the heme methyl resonances of cytochrome c' from Rhodospirillum rubrum (strain 1 DTCC) have been analyzed for the purpose of extracting information on the ligation states of the protein in both its oxidized and its reduced high spin forms. The methyl line widths in all protein forms were found to exhibit substantial quadratic field dependence consistent with significant contribution from Curie spin relaxation. For ferricytochrome c', the field-independent contribution to the methyl line width differed substantially between the acid-to-neutral pH form (I) and form II, stable in the pH range 9 to 11. Comparison of the calculated electron relaxation times indicates that form I has a weaker axial ligand field than either form II or aquometmyoglobin, which favors a five-coordinate I and a six-coordinate II. The appearance of two new resonances in the far downfield region upon converting I ..-->.. II, without the apparent loss of any hyperfine shifted signals from I, confirms the coordination of a sixth ligand in II. Qualitative comparisons of the line widths fo these two new signals to those of the heme methyls support an acidic residue as the sixth ligand. The detection of more » the characteristic exchangeable proton resonance of an axial imidazole and the importance of Curie spin relaxation in ferrocytochrome c' confirm its high-spin five-coordinated structure. 4 figures. « less