The Distal Pocket Histidine Residue in Horse Heart Myoglobin Directs the O-Binding Mode of Nitrite to the Heme Iron

Abstract

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 (NO2−) to nitric oxide (NO) under hypoxic conditions. We demonstrate here that the distal pocket histidine residue (His64) of horse heart metMbIII (i.e., ferric MbIII) has marked effects on the mode of nitrite ion coordination to the iron center. X-ray crystal structures were determined for the mutant proteins metMbIII H64V (2.0 Å resolution) and its nitrite ion adduct metMbIII H64V-nitrite (1.95 Å resolution), and metMbIII H64V/V67R (1.9 Å resolution) and its nitrite ion adduct metMbIII H64V/V67R-nitrite (2.0 Å resolution). These are compared to the known structures of wild type hh metMbIII and its nitrite ion adduct hh metMbIII-nitrite, which binds NO2− via an O-atom in a trans-FeONO configuration. Unlike wt metMbIII, no axial H2O is evident in either of the metMbIII 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 metMbIIIs with cysteine as a reducing agent and of the (ferrous) MbIIs with nitrite ion under anaerobic conditions. The MbIIs were generated by reduction of the MbIII 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 MbIIs 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.

Generating S-Nitrosothiols from Hemoglobin

“…In this sample, the addition of NO impacts both ferric populations but the nitrite MetHb population appears to decrease to a greater degree than the aquo-met population. tions of NO or NONOates but through an L-cysteine-induced reduction of ferric heme to ferrous heme (88). Any five coordinate deoxy-heme site (in Hb) can then generate NO from nitrite through the nitrite reductase reaction.…”

Combining selection valve and mixing chamber for nanoflow gradient generation: Toward developing a liquid chromatography cartridge coupled with mass spectrometer for protein and peptide analysis

“…Yi and RichterAddo's lab [13]. Briefly, gene engineered horse heart mutant myoglobin (hh Mb, H64V) was expressed in Escherichia coli, followed by ion-exchange chromatography purification.…”

“…It also removes nitrite ðNO 2 À Þ by reducing it to nitric oxide (NO) and lowers the energy status of heart cells, which subsequently prevents heat injury from hypoxic conditions [16,17]. It was reported that a single amino acid mutant of horse heart myoglobin (hh Mb, H64V) dramatically decreased the efficiency of hh Mb as a scavenger to remove NO 2 À , due to the mutation that changed the H-bonding in the distal pocket of the myoglobin [13]. Here, we coupled our nano-HPLC with a mass spectrometer (LCQ DECA XP plus, Thermo Fisher Scientific Inc., Waltham, MA) for protein separation and identification.…”

Mechanisms of Nitric Oxide Reactions Mediated by Biologically Relevant Metal Centers

“…However, there are other pathways to nitrosyl complexes. For example, nitrite reduction concomitant with (formal) oxidation of the metal can lead to a metal-nitrosyl complex illustrated in (16) [114][115][116][117].…”