Efficient Coupled Oxidation of Heme by an Active Site Variant of Horse Heart Myoglobin

Abstract

The coupled oxidation of protoheme IX at the active sites of heme proteins exposed to ascorbate in the presence of dioxygen has been known for over 65 years and is related to the catalytic conversion of protoheme IX to biliverdin by the enzyme heme oxygenase. The present report demonstrates that replacement of two residues present in the distal heme pocket of horse heart myoglobin (Mb), Val67 and Val68, with alanine and serine, respectively, increases the efficiency of coupled oxidation in the active site of myoglobin. HPLC analysis of the heme oxidation product demonstrates that the specificity of wild-type myoglobin for opening the heme ring at the α-meso carbon is retained by the variant. The infrared spectrum of the carbonyl derivative of the variant exhibits νCO bands of comparable intensity at 1948 and 1958 cm-1, which are consistent with the presence of the polar serine residue in close proximity to the bound ligand. The high-resolution crystal structure of the Val67Ala/Val68Ser metMb variant establishes that a hydrogen bond forms between the hydroxyl group of Ser68 and the coordinated water molecule. The increased rate of coupled oxidation exhibited by the variant is attributed to the increased polarity of the distal pocket created by the amino acid substitutions. These results are discussed in light of recent work concerning the active site of heme oxygenase.