The interaction between heme and protein in cytochrome c1.

The optical spectrum of reduced bovine cytochrome c1 at 77 K shows a fine splitting of the beta-band, which is indicative of the native conformation of the protein. At room temperature, this conformation is reflected in an absorbance band at 530 nm. The exposure of the heme of ferrocytochrome c1, investigated by means of solvent-perturbation spectroscopy, appears to be extremely sensitive to temperature and SH reagents bound to the oxidized protein. Addition of combinations of potential ligands to the isolated tryptic heme peptide of cytochrome c1 reveals that only a mixture of methionine and cysteine (or their equivalents) generates a beta-band at 77 K which is identical in shape to that of native cytochrome c1. In the EPR spectrum of a complex of ferrocytochrome c1 and nitric oxide at pH 10.5, no hyperfine splitting derived from a second ligated nitrogen atom could be detected. The results indicate that methionine and cysteine are the axial ligands of heme in cytochrome c1. The EPR spectrum of isolated ferricytochrome c1 is that of a low-spin heme iron compound with a gz value of 3.36 and a gy value of 2.04.