Abstract
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.
Highlights
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 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
The general appearance of this transform is typical of metalloporphyrin EXAFS.6
Summary
Table I) were prepared according to literature methods. The samples were ground pressed into and diluted with dry boron nitride pellets held inside an aluminum or LiBF4 and spacer. All EXAFS spectra of proteins were obtained at 2°C using sealed cells contained in a sample box flushed with helium. The most striking feature of this transform is that contributions are observed from atoms as far as 4.2 A (peak at 3.8 A) away from porphyrin EXAFS has shown that the assumption of a constant Eo the iron. This is quite different from the EXAFS observed for does not introduce a detectable inaccuracy in the determination of R, as long as the same Eo value is used for all compounds. The observation of such extended structure for P-450 is a reflection of the relative rigidity and high degree of
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