ELECTRON-SPIN ECHO ENVELOPE MODULATION STUDY OF OXYGENATED IRON-COBALT HYBRID HEMOGLOBINS REVEALS MOLECULAR-FEATURES ANALOGOUS TO THOSE OF THE OXY FERROUS PROTEIN

Two oxygenated iron-cobalt hybrid hemoglobins (Hbs), (alpha Co-O-2 bet a Fe-O-2)(2) and (alpha Fe-O-2 beta Co-O-2)(2), were studied by electr on spin echo envelope modulation (ESEEM) spectroscopy in order to meas ure (i) electron-nuclear hyperfine and nuclear quadrupole coupling to the N-epsilon of the proximal histidyl imidazole and (ii) nuclear hype rfine coupling to exchangeable H-2 in the oxyCo subunits. N-14 couplin gs were found to be smaller in the oxyCo alpha subunits than in the ox yCo beta subunits, suggesting a more ionic and shorter Co-O-2 bond in the a subunits [Lee et al. (1994) Biochemistry 33, 7609], which correl ates with the higher O-2 affinity found for (alpha Co beta Fe-O-2)(2) Hb than for (alpha Fe-O-2 beta Co)(2) Hb [Imai et al. (1980) J. Mol. B iol. 138, 635]. A smaller nuclear quadrupole coupling constant found f or the proximal histidyl N-epsilon in the oxyCo alpha subunits also su ggests an increase in the overlap between the N-epsilon sp(2) hybrid a nd the Co d(z)(2) orbital, i.e., a shorter Co-N-epsilon bond, than in the oxyCo beta subunits. On the other hand, the relative orientation o f the g and N-14(epsilon) nuclear quadrupole tensors, obtained by spec tral simulation, suggests that the Co-O-O bond angle is similar in the two types of oxyCo subunits. An X-ray crystallographic study of oxyFe Hb A [Shaanan, B. (1982) Nature 296, 683] has also reported similar F e-O-O bond angles in both alpha and beta subunits, but with shorter Fe -N-epsilon and Fe-O-2 bonds in the alpha subunits. A hyperfine-coupled H-2 was detected in the spectra of D2O-exchanged samples of both hybr id Hbs, although the resolution was only marginal in (alpha Fe-O-2 bet a Co-O-2)(2) Hb. These results support the idea of the presence of a h ydrogen bond to bound O-2 in both oxyCo alpha and beta subunits.