Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450_cam

Abstract

To investigate the functional and structural roles of the proximal thiolate ligand in cytochrome P450 cam , we prepared the C357H mutant of the enzyme in which the axial cysteine residue (Cys357) was replaced with a histidine residue. We obtained the unstable C357H mutant by developing a new preparation procedure involving in vitro folding of P450 cam from the inclusion bodies. The C357H mutant in the ferrous-CO form exhibited the Soret peak at 420 nm and the Fe-CO stretching line at 498 cm 21 , indicating a neutral histidine residue as the axial ligand. However, another internal ligand is coordinated to the heme iron as the sixth ligand in the ferric and ferrous forms of the C357H mutant, suggesting the collapse of the substratebinding site. The C357H mutant showed no catalytic activity for camphor hydroxylation and the reduced heterolytic/ homolytic ratio of the O±O bond scission in the reaction with cumene hydroperoxide. The present observations indicate that the thiolate coordination in P450 cam is important for the construction of the heme pocket and the heterolysis of the O±O bond.Keywords: P450; CPO; P420; oxygen activation; axial push effect.Heme-thiolate enzymes such as cytochrome P450 (P450) and chloroperoxidase from Caldariomyces fumago (CPO) activate heme-bound dioxygen and hydrogen peroxide and insert oxygen atoms into various hydrocarbons [1±4]. The enzymes have attracted much interest because the heterolytic O±O bond scission of the heme-bound dioxygen and the C±H bond activation of inactivated hydrocarbons are difficult in mild conditions [1,5]. As the enzymes have an anionic thiolate as the axial heme ligand [6,7], the strong electron donation of the thiolate is supposed to facilitate the heterolysis of the dioxygen that coordinates trans to the thiolate ligand [8,9]. The importance of the thiolate coordination for the activities of these enzymes is exemplified by their inactivated forms (P420 and C420) which lack the thiolate coordination [10,11]. However, the roles of Fe±S bond that are important to understand the molecular mechanism of the enzyme activities are still controversial [12,13].To clarify the roles of the thiolate coordination in the heme-thiolate enzymes, we replaced the axial histidine of myoglobin (Mb) with cysteine (H93C), and investigated its catalytic reactivities [14,15]. We observed that the thiolate ligand of Mb H93C accelerated the heterolysis of the O±O bond of cumene hydroperoxide and enhanced the hydrogen peroxide supported epoxidation of styrene [15]. These observations allowed us to propose that the thiolate ligation in heme proteins encourages the heterolytic cleavage of the O±O bond [15]. Our proposal is further confirmed by Higuchi et al. who demonstrated that the ligation of alkyl thiolate to iron porphyrin facilitates the O±O bond heterolysis of alkyl peracid in hydrophobic solvent [16,17]. These model works indicate the importance of the thiolate coordination in the heterolytic O±O bond scission.We have also shown experimentally that the thiolate ligand control...

Redox and Chemical Activities of the Hemes in the Sulfur Oxidation Pathway Enzyme SoxAX

“…The change in intensity observed at ϳ550 nm is typically much larger when thiolate-ligated ferric heme is reduced to ferrous thiol-, rather than thiolate-, ligated heme (33)(34)(35) because the absorbance band of the porphyrin -* transition is much sharper in the former case. The intensity of the sharp features in this region of both the MCD and the electronic absorbance spectra of SoxAX clearly increases at low potential, with the best fit to the data being obtained when these changes are attributed to the heme with the lowest midpoint potential.…”

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

“…It has been difficult, however, to modulate its properties as an electron donor without destroying the heme environment. For example, mutating the proximal cysteine to histidine, a residue often found in the corresponding position of non-P450-type heme proteins, inactivates P450s (4,5). Consequently, more conservative replacement of cysteine by a nonstandard amino acid such as selenocysteine is attractive.…”

The Influence of the Proximal Thiolate Ligand and Hydrogen Bond Network of the Proximal Helix on the Structural and Biochemical Properties of Chloroperoxidase

“…(9). Several studies utilized heme Soret band absorption location for a ferrous protein-CO complex to investigate the nature of the proximal ligand, stating that cysteine and histidine, upon binding CO, would produce the peak near 450 nm and 420 nm, respectively (106,196,(211)(212)(213)(214)(215).…”

“…Furthermore, replacement of the proximal Cys with a His in P450s results in a complete loss of oxygenase activity (212)(213)(214). However, similar substitution in CPO results in only slightly decreased epoxidation rates (215).…”