Engineering myoglobin into both a structural and functional model of nitric oxide reductase

Nitric oxide reductase (NOR) is the only metalloenzyme in the denitrification pathway for which there is no crystal or solution structure. NORs are structurally homologous to subunit I of HCOs (heme-copper oxidases); however, the copper site (CuB) is replaced with a non-heme iron site (FeB) in NOR.1-3 There are several conserved glutamates in NOR that are not found in HCO.4,5 A glutamate is predicted to play a role in iron binding to the FeB site and/or catalysis. In addition to their structural similarities, NOR and HCO also have cross-reactivity. NOR is capable of O2 reduction and some members of the HCO superfamily are capable of NO reduction.6-8 Therefore, an important question is whether the identity of the metal helps tune activity in homologous protein metal binding sites, namely the CuB site in HCO and the Fe B site in NOR.;Our group has engineered a CuB site into the scaffold protein myoglobin (CuBMb, swMb L29H, F43H, H64; swMb is sperm whale myoglobin). 9 Copper-bound CuBMb catalytically reduces NO to N 2O at a rate of ∼2 mol NO·mol CuBMb-1 ·min-1, making CuBMb a functional model of NOR.10 UV-vis and EPR studies support the weakening, but not cleavage, of the proximal histidine bond to the heme iron upon NO binding, resulting in an intact six coordinate heme-NO species. A six coordinate species was also observed for the low activity ba3 oxidases.11 In contrast, a five coordinate heme-NO intermediate was observed for NOR.12,13 Therefore, these results suggest that a five coordinate heme-NO species is essential for high NO activity.;To create a closer structural model of the heme/FeB site and to mimic the conserved glutamate(s) in NOR, a glutamate was introduced near the heme of CuBMb, called FeBMb (swMb L29H, F43H, H64, V68E). Crystallographic and/or spectroscopic studies show that FeBMb can bind Fe or Cu in its engineered FeB site. From the crystal structure of Fe(II)-FeBMb, the Fe···Fe distance of 4.65 Å and Fe-O-Fe angle of 138° (O represents an oxygen atom). The FeB ion binds to all three His of the engineered metal binding site, one water molecule, and one O atom of the Glu68.;From GC/MS and UV-vis studies, FeBMb was found to reduce NO to N2O in the presence of Fe(II) or Cu(I) under one turnover conditions. However, NO activity is lower with Fe than with Cu bound to the same protein scaffold. In contrast, CuBMb, which does not contain a glutamate ligand near the heme, showed no evidence of either Fe binding or NO activity with Fe. These results support the vital role that the glutamate plays in retaining the iron ion in the FeB site.;The introduction of a glutamate in close proximity to the heme (i.e., FeBMb, E1/2 = -158 mV) appears to lower the reduction potential of the heme by >200 mV, when compared to CuBMb (E 1/2 = +77 mV), which does not contain a glutamate ligand. The modulation of the heme potential by a glutamate also mimics that of NOR, where the catalytic heme reduction potential was found to be much lower (>200 mV) than the surrounding cofactors.;The low reduction potential of FeBMb (-158 mV ± 4 vs. NHE) requires it to be reduced by a strong reductant. To study the NO reduction activity of FeBMb under catalytic conditions, it is necessary to find a reductant that can rapidly reduce FeBMb, while minimizing the non-enzymatic reduction of NO to N2O, a reaction that is more thermodynamically favorable than protein reduction. A variety of reductants were investigated, but none of them satisfied the above criteria.;To date, FeBMb is the first protein model of the FeB site of NOR and provides the first evidence, from an NOR model, that the identity of the metal in analogous non-heme metal binding sites of HCO (i.e., CuB) and NOR (i.e., FeB) can tune NO activity. In summary, FeBMb is both a structural and functional model of NOR. (Abstract shortened by UMI.).