Influence of the heme distal pocket on nitrite binding orientation and reactivity in Sperm Whale myoglobin

• Published in: Biochemical Journal
• Main Authors: Tse, Wilford, Whitmore, Nathan, Cheesman, Myles R., Watmough, Nicholas J.
• Format: Text
• Language: English
• Published: Portland Press Ltd. 2021
• Subjects: Biophysics; Sperm whale
• Online Access: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925009/; http://www.ncbi.nlm.nih.gov/pubmed/33543749; https://doi.org/10.1042/BCJ20200596

Nitrite binding to recombinant wild-type Sperm Whale myoglobin (SWMb) was studied using a combination of spectroscopic methods including room-temperature magnetic circular dichroism. These revealed that the reactive species is free nitrous acid and the product of the reaction contains a nitrite ion bound to the ferric heme iron in the nitrito- (O-bound) orientation. This exists in a thermal equilibrium with a low-spin ground state and a high-spin excited state and is spectroscopically distinct from the purely low-spin nitro- (N-bound) species observed in the H64V SWMb variant. Substitution of the proximal heme ligand, histidine-93, with lysine yields a novel form of myoglobin (H93K) with enhanced reactivity towards nitrite. The nitrito-mode of binding to the ferric heme iron is retained in the H93K variant again as a thermal equilibrium of spin-states. This proximal substitution influences the heme distal pocket causing the pK(a) of the alkaline transition to be lowered relative to wild-type SWMb. This change in the environment of the distal pocket coupled with nitrito-binding is the most likely explanation for the 8-fold increase in the rate of nitrite reduction by H93K relative to WT SWMb.