Sulfur Donor Ligand Binding to Ferric Cytochrome P - 450 - CAM and

The binding of thiol, thiolate, thioether, and disulfide sulfur donor ligands to ferric cytochrome P-450-CAM and myoglobin has been investigated by W-visible absorption, magnetic circular dichroism (MCD), and EPR spectroscopy. For ferric P-450, the binding of all sulfur donors is competitive with substrate binding. Addition of thiols to P-450 leads to interconvertible thiol or thiolate-bound species depending on the thiol acidity (pa) and the solution pH; ligation of thiols lowers their pK, by about 4 units. In contrast, only the thiolate-bound form is seen for myoglobin regardless of thiol acidity or solution pH (5.5-ll.O), indicating that the heme iron of myoglobin is less electron-rich than that of P-450. Thiolate ligands show much higher affinity (& lob6 M) for ferric P-450 than do thiols (& M). The affinity of thioethers for P-450 (a M) is pH-independent (pH 5.5-9.0). The observed disulfide coordination to P-450 represents the first example of disulfide ligation to heme iron; no significant evidence for thioether or disulfide binding to myoglobin is seen. Except for the thiolate complexes, the W-visible and MCD spectral properties of the other sulfur donor P-450 complexes are similar to, although distinguishable from, those of native P-450. The ferric P-450. thiolate complexes exhibit MCD spectra resembling that of ferrous P-450-CO; both also exhibit unique hyperporphyrin (split Soret) W-visible spectra. The EPR spectra of all P-450 complexes examined display very narrow spread g-values such as are characteristic of native P-450, indicating that the endogenous cysteinate axial ligand is retained upon complex formation. The dissimilarities observed between P-450 and myoglobin in their reactivity toward sulfur donor ligands at least partly reflect the variation in heme iron electron density resulting from their different endogenous axial ligands and may, in turn, help to explain their respective physiological functions of oxygen activation and reversible oxygen binding.