Selenolate Complexes of CYP101 and the Heme-Bound hHO-1/H25A Proximal Cavity Mutant

Abstract

Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-visible spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenolate ligands also bound to this cavity mutant under anaerobic conditions, but reduced the heme iron to the ferrous state as shown by formation of a ferrous-CO complex. Under aerobic conditions, the selenolate but not thiolate ligand was rapidly oxidized. These results indicate that selenocysteine-coordinated heme proteins will not be stable species in the absence of a redox potential stabilizing effect.Cytochrome P450 enzymes are a superfamily of heme proteins that employ a cysteine thiolate as the proximal ligand to the heme. 1 The P450-catalyzed reaction has been thought for three decades to be mediated by a Compound I-like ferryl species that is generated by reductive activation of molecular oxygen at the prosthetic heme iron center. Despite support by a variety of data for a Compund I-like ferryl species in the P450 catalytic cycle, it has remained elusive towards spectroscopic detection. 2,3 Conflicting results have also been obtained from determinations of the lifetime of the ferryl species using radical clock substrates. 4 Direct detection and characterization of the reactive oxidizing species in P450 enzymes would therefore be highly desirable. Recent computational studies have suggested that a seleno-CYP101 mutant in which the cysteine ligand is replaced by selenocysteine might provide an approach to investigation of the elusive P450 oxidizing species. 5 The Se-Compound I intermediate was predicted to form faster than the wild-type S-Compound I, and to be consumed more slowly. The increased lifetime of the intermediate suggested by the calculations indicated that it might become spectroscopically detectable.Targeted insertion of selenocysteine into a protein sequence through genetic code manipulations is a complex cotranslational process not easily adapted to P450 enzymes. 6,7 An alternative and straightforward approach to investigate the effect of ligands in heme proteins has been to use a cavity mutant in which the proximal amino acid ligand is replaced by a small and noncoordinating amino acid such as an alanine or glycine, creating a pocket that can accommodate exogenous unnatural ligands. A successful example of this is provided by the sperm whale myoglobin (Mb) H93G mutant, which has been extensively used for this purpose. 8,9 The crystal structures of β-mercaptoethanol and acetate-bound Mb H93G have recently confirmed that the exogenous ligands bind in the proximal ligation site.

Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine

“…4B). Complexes of WT P450cam with benzeneselenol show a similar red shift relative to the corresponding thiophenol complexes (35), consistent with the lower ionization energy of selenium.…”

“…Selenolate ligands like benzeneselenol, bound to the proximal cavity of a mutant P450-like enzyme lacking a distal electrondonating substituent, have been shown to reduce the heme iron to the ferrous state (35). In contrast, the ferric form of C357U P450cam* is stable under aerobic conditions.…”

Covalent heme attachment to the protein in human heme oxygenase-1 with selenocysteine replacing the His25 proximal iron ligand

“…We recently reported formation of a complex of the proximal hHO-1 H25A cavity mutant with an external benzeneselenolate anion as the iron ligand (37). Interestingly, in this complex, the selenium transfers an electron to the heme iron atom, as demonstrated by partial formation of a ferrous-CO complex in the absence of other reducing agents.…”

The broad diversity of heme-protein cross-links: An overview

“…It is interesting to find that when the heme axial ligand His25 was replaced by a SeCys, SeCys25 formed a covalent bond (C-Se bond) with a heme vinyl group under reducing conditions, which was not observed for the H25C mutant with an axial ligand Cys. Based on the fact that external selenolate ligand can transfer an electron to the heme iron [62], the authors proposed a mechanism for formation of the novel C-Se bond, which involves generation of a selenyl radical and its addition to a heme vinyl group (Scheme 3).…”