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

Wilford Tse, Nathan Whitmore, Myles R. Cheesman, Nicholas J. Watmough

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)
8 Downloads (Pure)


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 pKa 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.
Original languageEnglish
Pages (from-to)927-942
Number of pages16
JournalBiochemical Journal
Issue number4
Publication statusPublished - 26 Feb 2021

Cite this