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

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.

Nitrophorins and nitrobindins: structure and function

Classical all α-helical globins are present in all living organisms and are ordered in three lineages: (i) flavohemoglobins and single domain globins, (ii) protoglobins and globin coupled sensors and (iii) truncated hemoglobins, displaying the 3/3 or the 2/2 all α-helical fold. However, over the last two decades, all β-barrel and mixed α-helical-β-barrel heme-proteins displaying heme-based functional properties (e.g. ligand binding, transport and sensing) closely similar to those of all α-helical globins have been reported. Monomeric nitrophorins (NPs) and α1-microglobulin (α1-m), belonging to the lipocalin superfamily and nitrobindins (Nbs) represent prototypical heme-proteins displaying the all β-barrel and mixed α-helical-β-barrel folds. NPs are confined to the Reduviidae and Cimicidae families of Heteroptera, whereas α1-m and Nbs constitute heme-protein families spanning bacteria to Homo sapiens. The structural organization and the reactivity of the stable ferric solvent-exposed heme-Fe atom suggest that NPs and Nbs are devoted to NO transport, storage and sensing, whereas Hs-α1-m participates in heme metabolism. Here, the structural and functional properties of NPs and Nbs are reviewed in parallel with those of sperm whale myoglobin, which is generally taken as the prototype of monomeric globins.

Myoglobin-catalyzed intermolecular carbene N–H insertion with arylamine substrates

Engineered variants of sperm whale myoglobin efficiently catalyze the insertion of α-diazo esters into the N–H bond of arylamines, featuring a combination of high chemoselectivity, elevated turnover numbers, and broad substrate scope.