Q-band ENDOR (electron nuclear double resonance) of the heme o3 liganding environment at the binuclear center in cytochrome bo3 from Escherichia coli

The liganding environment and electronic structure of high-spin ferric heme o3 in the binuclear center of bo3 ubiquinol oxidase were probed with Q-band (34.1 GHz) ENDOR. We studied forms of the enzyme where reduction eliminated antiferromagnetic coupling to the nearby Cu(B) center. ENDOR comparisons were made to ¹⁴N heme and histidine nitrogen features, to exchangeable proton features, and to the ¹⁷O-water feature of aquometmyoglobin, a high-spin ferric heme protein with a known axial water ligand. Nitrogen features observed from heme and proximal histidine of cytochrome o3 occurred in the range of frequencies where they had previously been observed for aquometmyoglobin. However, the proximal histidine of cytochrome o3 was notable in revealing more disorder and a wider range in its hyperfine couplings than in aquometmyoglobin. Di-oxygen-induced turnover of the bo3 enzyme altered both the heme and histidine electronic structure so as to show after turnover a simpler, better resolved heme and histidine pattern with greater similarity to the pattern found in aquometmyoglobin. We saw no evidence from cytochrome o3 for the 6 MHz exchangeable water proton coupling and the 17.5 MHz ¹⁷O-water coupling exhibited by aquomet-myoglobin. A plausible conclusion from such a negative result is that the high-spin ferric o3 heme which we studied has no covalently attached axial sixth OH(X) ligand when magnetically decoupled from Cu(B). Comparison of cytochrome o3 in protonated and deuterated solvents definitively indicated no exchangeable proton couplings greater than 3.5 MHz. An implication of our study is that in the magnetically decoupled high-spin ferric cytochrome o3 there is either no sixth OH(X) ligand or, if there is any 'sixth' OH(X) ligand to cytochrome o3 that can exchange with ¹⁷O-water, it would have to be off-axis, disordered, and weakly liganded to the heme.