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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 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.

SUBMITTER: Tse W

PROVIDER: S-EPMC7925009 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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

Tse Wilford W Whitmore Nathan N Cheesman Myles R MR Watmough Nicholas J NJ

The Biochemical journal 20210201 4

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 f ...[more]

PMID: 33543749

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Project description:It is now well-established that mammalian heme proteins are reactive with various nitrogen oxide species and that these reactions may play significant roles in mammalian physiology. For example, the ferrous heme protein myoglobin (Mb) has been shown to reduce nitrite (NO(2)(-)) to nitric oxide (NO) under hypoxic conditions. We demonstrate here that the distal pocket histidine residue (His64) of horse heart metMb(III) (i.e., ferric Mb(III)) has marked effects on the mode of nitrite ion coordination to the iron center. X-ray crystal structures were determined for the mutant proteins metMb(III) H64V (2.0 A resolution) and its nitrite ion adduct metMb(III) H64V-nitrite (1.95 A resolution), and metMb(III) H64V/V67R (1.9 A resolution) and its nitrite ion adduct metMb(III) H64V/V67R-nitrite (2.0 A resolution). These are compared to the known structures of wild-type (wt) hh metMb(III) and its nitrite ion adduct hh metMb(III)-nitrite, which binds NO(2)(-) via an O-atom in a trans-FeONO configuration. Unlike wt metMb(III), no axial H(2)O is evident in either of the metMb(III) mutant structures. In the ferric H64V-nitrite structure, replacement of the distal His residue with Val alters the binding mode of nitrite from the nitrito (O-binding) form in the wild-type protein to a weakly bound nitro (N-binding) form. Reintroducing a H-bonding residue in the H64V/V67R double mutant restores the O-binding mode of nitrite. We have also examined the effects of these mutations on reactivities of the metMb(III)s with cysteine as a reducing agent and of the (ferrous) Mb(II)s with nitrite ion under anaerobic conditions. The Mb(II)s were generated by reduction of the Mb(III) precursors in a second-order reaction with cysteine, the rate constants for this step following the order H64V/V67R > H64V >> wt. The rate constants for the oxidation of the Mb(II)s by nitrite (giving NO as the other product) follow the order wt > H64V/V67R >> H64V and suggest a significant role of the distal pocket H-bonding residue in nitrite reduction.

Project description:Heme nitrite reductases reduce NO2- by 1e-/2H+ to NO or by 6e-/8H+ to NH4+ which are key steps in the global nitrogen cycle. Second-sphere residues, such as arginine (with a guanidine head group), are proposed to play a key role in the reaction by assisting substrate binding and hydrogen bonding and by providing protons to the active site for the reaction. The reactivity of an iron porphyrin with a NO2- covalently attached to a guanidinium arm in its 2nd sphere was investigated to understand the role of arginine residues in the 2nd sphere of heme nitrite reductases. The presence of the guanidinium residue allows the synthetic ferrous porphyrin to reduce NO2- and produce a ferrous nitrosyl species ({FeNO}7), where the required protons are provided by the guanidinium group in the 2nd sphere. However, in the presence of additional proton sources in solution, the reaction of ferrous porphyrin with NO2- results in the formation of ferric porphyrin and the release of NO. Spectroscopic and kinetic data indicated that re-protonation of the guanidine group in the 2nd sphere by an external proton source causes NO to dissociate from a ferric nitrosyl species ({FeNO}6) at rates similar to those observed for enzymatic sites. This re-protonation of the guanidine group mimics the proton recharge mechanism in the active site of NiR. DFT calculations indicated that the lability of the Fe-NO bond in the {FeNO}6 species is derived from the greater binding affinity of anions (e.g. NO2-) to the ferric center relative to neutral NO due to hydrogen bonding and electrostatic interaction of these bound anions with the protonated guanidium group in the 2nd sphere. The reduced {FeNO}7 species, once formed, is not affected significantly by the re-protonation of the guanidine residue. These results provide direct insight into the role of the 2nd sphere arginine residue present in the active sites of heme-based NiRs in determining the fate of NO2- reduction. Specifically, the findings using the synthetic model suggest that rapid re-protonation of these arginine residues may trigger the dissociation of NO from the {FeNO}6, which may also be the case in the protein active site.

Dataset Information

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

Publications

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

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