The Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin

摘要

The proximal side of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata has been modified via site-directed mutagenesis of methionine-86 into aspartate (M86D) to introduce an Asp-His-Fe triad charge relay. X-ray crystallographic structure determination of the metcyano forms of M86D (PDB 3MYN) and M86E (PDB 3MYM) mutants reveals the structural origins of a stable catalytic triad in DHP A. A decrease in rate of H2O2 activation, as well as a lowered reduction potential than the wild-type enzyme was observed in M86D. One possible explanation for the significantly lower activity is an increased affinity for the distal histidine to bind to the heme Fe to form a bis-histidine adduct. Resonance Raman spectroscopy demonstrates a pH-dependent ligation by the distal histidine in M86D, which is indicative of an increased trans effect. At pH 5.0, the heme Fe is 5-coordinate and this resembles the wild-type DHP A resting state. However, at pH 7.0, the distal histidine appears to form a 6-coordinate ferric bis-histidine (hemichrome) adduct. These observations can be explained by the effect of the increased positive charge on the heme Fe on the formation of a 6-coordinate low-spin adduct, which inhibits the ligation and activation of H2O2 as required for peroxidase activity. The results suggest that the role of the proximal charge relay in peroxidases regulates the redox potential of the heme Fe, but that the trans effect is a carefully balanced property that can both activate H2O2 and attract ligation by the distal histidine. To understand the balance of forces that modulate peroxidase reactivity, three mutants in the M86 position, M86A, M86D, and M86E were studied by spectroelectrochemistry and NMR spectroscopy of ¹³C¹⁵N -labeled cyanide adducts as probes of the redox potential and of the trans effect in the heme Fe, both of which can be correlated with the proximity of negative charge to the Nδ hydrogen of the proximal histidine, consistent with an Asp-His-Fe charge relay observed in heme peroxidases.