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 R-meso carbon is retained by the variant. The infrared spectrum of the carbonyl derivative of the variant exhibits ν CO 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.

Heme oxygenase, steering dioxygen activation toward heme hydroxylation

“…Hence, the fact that the oxidation of heme by HO and by the coupled oxidation process need O 2 and reducing equivalents and proceed via the formation of the same intermediates (meso-hydroxyheme and verdoheme), suggested that the mechanisms of these processes may have been the same. Consequently, the coupled oxidation process has been used to model the process of heme oxidation conducted by the enzyme heme oxygenase [122,125,[127][128][129][130][131][132][133][134][135]. As will be discussed in more detail below, however, recent work has demonstrated that the two processes differ fundamentally in the nature in which the oxidant is generated [136,137].…”

Myoglobin/gold nanoparticles/carbon spheres 3-D architecture for the fabrication of a novel biosensor

“…Myoglobin (Mb) is a single-chain protein of 153 amino acids containing a heme (iron-containing porphyrin) group in the center which is found in mammalian skeleton and muscle tissues. Mb functions physiologically in the storage of oxygen and in the enhancement of the rate of oxygen diffusion, and has catalytic activity for H 2 O 2 decomposition similar to horseradish peroxidase [17,18]. It is an ideal model molecule for the study of electron transfer reactions of heme proteins and also for biosensing and biocatalysis.…”

A Novel NH2/ITO Ion Implantation Electrode: Preparation, Characterization, and Application in Bioelectrochemistry

“…Myoglobin (Mb) is a single-chain protein of 153 amino acids, containing a heme (iron-containing porphyrin) group in the center. Mb is found in mammalian skeleton and muscle tissues, which functions in the storage of oxygen and in the enhancement of the rate of oxygen diffusion [1,2]. But the electron transfer of Mb is very slow on conventional electrodes because of protein adsorption and subsequent passivation of the electrodes surface.…”