Sulfur donor ligand binding to ferric cytochrome P-450-CAM and myoglobin. Ultraviolet-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopic investigation of the complexes.

M Sono,L A Andersson,J H Dawson

doi:10.1016/s0021-9258(18)34332-1

Abstract

The binding of thiol, thiolate, thioether, and disulfide sulfur donor ligands to ferric cytochrome P-450-CAM and myoglobin has been investigated by UV-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 (pKa) and the solution ph; ligation of thiols lowers their pKa by about 4 units. In contrast, only the thiolate-bound form is seen for myoglobin regardless of thiol acidity or solution pH (5.5-11.0), indicating that the heme iron of myoglobin is less electron-rich than that of P-450. Thiolate ligands show much higher affinity (Kd approximately 10(-6) M) for ferric P-450 than do thiols (Kd approximately 10(-3) M). The affinity of thioethers for P-450 (Kd approximately 10(-3) 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 UV-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) UV-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.

Highlights

The binding of thiol, thiolate, thioether, and disulfide substrates [2,3,4]
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
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

Summary

RESULTS

Ferric P-450 Thiolate Complexes-Titration of either substrate-free, six-coordinate, low spin, or substrate-bound, fivecoordinate, high spin P-450 with p-chlorothiophenol (Fig. 1) resultsina complex with ahyperporphyrin(split Soret) spectrum essentially identical with that of a syntheticbisthiolate heme iron model complex previously reported by Ruf and Wende [15]. Ferric P-450 Thioether and Disulfide Complexes--In Fig. 3, A and B, MCD and UV-visible spectra of native, ligandfree P-450 and its complexes with dimethyl sulfide and dimethyl disulfide are overplotted. In the MCD spectrum (Fig. 4A), at high pH only a single broad derivative-shaped feature is seen while a t low pH a three-bandpattern (peaks at -460 nm and -515 nm and a derivative-shaped curve centered a t 563 nm) is seen. The pH-dependent absorption spectrum of the 1-propanethiol.P-450 complex does not share isosbestic points with the free enzyme as is apparent when one examines the spectra aroundthe a-peak(569nm) of the latter(Fig.4B). The CD spectrum of the P-450 hydrogen sulfide complex (Fig. 5A) is exceptional and obviously distinct from those of theother ligand complexes examined (Fig. 5, B-D) The former exhibitsonly positive features between300 and 700 nm with relatively strong Soret andvisible peak intensity. The othceormplexes show mostly negative signals in the Soretregion withtrough intensities abouhtalf the peak c 24

Ferric P-450 plus 1- Propanethiol I N SHI

DISCUSSION

CONCLUSION

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