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

2009; American Chemical Society; Volume: 131; Issue: 50 Linguagem: Inglês

10.1021/ja904726q

1943-2984

Jun Yi, Julie L. Heinecke, Hui Tan, Peter C. Ford, George B. Richter‐Addo,

Neonatal Health and Biochemistry

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 (wt) 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.