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

Abstract

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 environment of the distal pocket coupled with nitrito-binding is the most likely explanation for the eight-fold increase in the rate of nitrite reduction by H93K relative to WT SWMb.

Nature's nitrite-to-ammonia expressway, with no stop at dinitrogen

“…The scaffold provided a remarkably versatile template for the preparation of model heme complexes and was used to mimic the heme iron coordination structure of native heme proteins, such as NrfA (Fe-N Lys ), the CooA transcription factor (Fe-N Pro ), or cytochrome f (Fe-N Tyr ) [ 226 , 227 ]. Along these lines, Nicholas Watmough and associates (University of East Anglia) created the His93Lys variant of sperm whale myoglobin and studied its binding and reactivity with nitrite [ 228 ]. Substitution of the proximal His ligand with Lys led to an eightfold increase in the rate of NO 2 − → NO reduction relative to wild-type myoglobin.…”

Noncovalent π Interactions in Mutated Aquomet-Myoglobin Proteins: A QM/MM and Local Vibrational Mode Study

“…Caused by this arrangement, there is only enough space for small signaling diatomic ligands such as O 2 , NO, CN, and CO to enter the active site and potentially bind to iron. In the ferrous form of Mb, the diatomic ligands reversibly bind to Fe initializing different biological functions, − which has led to an increased interest in quantifying Fe–ligand bonding in these complexes. , The effect of water binding to the active site of ferric Mb (labeled as aquomet-Mb) has been another target of interest because it allows us to illuminate the dual role of the distal histidine (H64) in stabilizing ligand binding at the heme and controlling access to the protein interior . Besides altering the ligand of the active site, others have modified the Mb protein to study a variety of different properties such as metal–ligand interactions (typically through resonance Raman techniques), protein function, and dynamics …”

Reversible thermally induced spin crossover in the myoglobin–nitrito adduct directly monitored by resonance Raman spectroscopy

“…The ferric heme Fe-ONO adduct has been assigned to low spin (LS) or high spin (HS) or LS/HS equilibrium states by different research groups that employed various spectroscopic methods including cryo EPR, room temperature resonance Raman and MCD spectroscopies as well as theoretical calculations. [20][21][22][23][24][25] The spin state of heme complexes is closely related to their reactivity and thus important in dening the course of biochemical reactions. In fact, one should consider that ligand/ substrate binding to the heme Fe and/or electron transfer reactions are frequently accompanied by spin transitions that control heme-protein functions and mechanisms of enzymatic reactions.…”

“…The ferric heme Fe–ONO adduct has been assigned to low spin (LS) or high spin (HS) or LS/HS equilibrium states by different research groups that employed various spectroscopic methods including cryo EPR, room temperature resonance Raman and MCD spectroscopies as well as theoretical calculations. 20–25…”