Endogenous cysteine ligation in ferric and ferrous cytochrome P-450. Direct evidence from x-ray absorption spectroscopy.

Extended x-ray absorption fine structure spectroscopy has been applied to the elucidation of the structure of the heme iron site of bacterial cytochrome P-450. The low spin ferric, high spin ferric, ferrous, and ferrous carbonyl states of the enzyme have been examined. Curve-fitting analysis of the data provides direct and compelling evidence for the presence of a sulfur atom in the first coordination sphere of the iron. The iron-nitrogen (porphyrin) distances indicate five coordination in high spin ferric and ferrous P-450 and six coordination in low spin ferric and ferrous carbonyl P-450. The iron-sulfur distances are consistent with thiolate ligation, presumably from cysteinate, in all four states of the enzyme. In each case, the iron-sulfur bond distance is equal to or shorter than the analogous Fe-S bonds in model iron porphyrin thiolate complexes whose crystal structures have been determined. Since known thiol-sulfur:iron-heme bond distances are noticeably longer than the corresponding thiolate bonds, the X-ray absorption fine structure results strongly suggest that, in each P-450 state examined, the sulfur donor is a thiolate. The results reported in this paper concerning the ligand identity, state of protonation, and metal-ligand bond distances are of critical importance to a complete description of the P-450 reaction cycle and its mechanism of oxygen activation.