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

Abstract

The unique monooxygenase activity of cytochrome P450cam has been attributed to coordination of a cysteine thiolate to the heme cofactor. To investigate this interaction, we replaced cysteine with the more electron-donating selenocysteine. Good yields of the selenoenzyme were obtained by bacterial expression of an engineered gene containing the requisite UGA codon for selenocysteine and a simplified yet functional selenocysteine insertion sequence (SECIS). The sulfur-to-selenium substitution subtly modulates the structural, electronic, and catalytic properties of the enzyme. Catalytic activity decreases only 2-fold, whereas substrate oxidation becomes partially uncoupled from electron transfer, implying a more complex role for the axial ligand than generally assumed.

Fig. 1.

The reaction path of P450cam. Compound I, a ferryl-oxo π-cation porphyrin radical, is the putative oxidant that reacts directly with substrate. Dashed arrows indicate possible uncoupling processes.

Fig. 2.

Design of a simplified SECIS element for P450cam. (A) Canonical SECIS element from E. coli formate dehydrogenase as encoded by the fdhH gene. The boxed region shows the minimal SECIS element with essential SelB-contacting bases circled. (B) Sequence of the P450cam gene immediately downstream of the codon for the proximal heme ligand, Cys-357. Sequence differences relative to the canonical SECIS element are highlighted in blue. (C) Simplified SECIS element for production of C357U P450cam*, with the mutations that were introduced shown in red. (D) Gene and amino acid sequences of WT P450cam, the control protein P450cam*, which has a proximal cysteine ligand in addition to the SECIS mutations, and C357U P450cam*. Mutations are shown in red.

Fig. 3.

Structure of C357U P450cam* in the camphor-bound state. (A) Active site of the selenoenzyme (yellow) superimposed on that of P450cam* (green) and WT P450cam (1dz4, gray); all 3 structures were determined from the monoclinic crystal form. Although the axial cysteine ligand at position 357 was substituted by selenocysteine, the structural integrity of the enzyme is maintained. The increased Fe-Se distance may account for the 2 side chain conformations observed for Leu-358 in the selenoenzyme. The camphor-binding site is not affected by the mutations (see Fig. S2). The camphor molecule has been omitted for clarity. (B) View of the region around Gln-366 in C357U P450cam* (yellow), P450cam* (green), and the WT protein (gray). Mutating the native glutamate residue to glutamine induces displacement of the connected chain of ordered water molecules.

Fig. 4.

Characterization of C357U P450cam* (red) and the control protein P450cam* (black). (A) UV-vis absorption spectra of the ferric resting state with bound camphor. (B) Ferrous CO form of the enzymes. (C) EPR spectra at 15 K. (D) Transformation of camphor by C357U P450cam* at 25 °C as a function of time.