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-ban d (34.1 GHz) ENDOR. We studied Forms of the enzyme where reduction eliminat ed antiferromagnetic coupling to the nearby Cu-B center. ENDOR comparisons were made to N-14 heme and histidine nitrogen features, to exchangeable pro ton features, and to the O-17-water feature of aquometmyoglobin, a high-spi n ferric heme protein with a known axial water ligand. Nitrogen features ob served from heme and proximal histidine of cytochrome o3 occurred in the ra nge of frequencies where they had previously been observed for aquometmyogl obin. However, the proximal histidine of cytochrome o3 was notable in revea ling more disorder and a wider range in its hyperfine couplings than in aqu ometmyoglobin. Di-oxygen-induced turnover of the bo3 enzyme altered both th e heme and histidine electronic structure so as to show after turnover a si mpler, better resolved heme and histidine pattern with greater similarity t o 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 O-17- water coupling exhibited by aquometmyoglobin. A plausible conclusion from s uch a negative result is that the high-spin Ferric o3 heme which we studied has no covalently attached axial sixth OHx ligand when magnetically decoup led from Cu-B. comparison of cytochrome o3 in protonated and deuterated sol vents 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 OHx ligand or, if t here is any "sixth" OHx ligand to cytochrome o3 that can exchange with O-17 -water, it would have to be off-axis, disordered, and weakly liganded to th e heme.