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 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.
Highlights
Heme containing nitrite reductases play a significant role in the transformation of nitrite to both nitric oxide and ammonia in animals, plants and bacteria
What is the active form of the nitrite ion in solution and is the product of its reaction with myoglobin the same nitrito-bound form observed in crystallographic experiments? Secondly does substitution of the proximal heme ligand (His-93) of myoglobin with lysine have any consequences for the reactivity of the protein towards nitrite or the mode of ligand binding? Here we describe the expression and characterisation of a novel form (H93K) of Sperm Whale myoglobin (SWMb) and show for the first time that room-temperature magnetic circular dichroism (RTMCD) spectroscopy reliably discriminates between the nitrito- and nitro-modes of binding to heme iron
We establish that WT SWMb reacts rapidly with free nitrous acid to form a stable product that has a RT-Magnetic Circular Dichroism (MCD) spectrum containing signals that arise from both high- and low-spin hemes
Summary
Heme containing nitrite reductases play a significant role in the transformation of nitrite to both nitric oxide and ammonia in animals, plants and bacteria. After addition of 100 mM nitrite to WT SWMb all bands in the UV-visible region MCD spectrum remain consistent with a Ferric heme species (Supplementary Figure S1A); none of the heme becomes reduced, but the bisignate Soret feature in the MCD spectrum shifts from 409 nm to 415 nm and increases in ( peak-to-trough) intensity to ≈40 M−1 cm−1 T−1 indicating a sub-stoichiometric high-spin to low-spin conversion (Supplementary Figure S1B).
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