EPR studies of the dynamics of rotation of dioxygen in model cobalt(II) hemes and cobalt-containing hybrid hemoglobins

Earlier we showed that the shapes of the EPR spectra of cobalt(II) porphyrinate(nitrogen base)(dioxygen) complexes in fluid solution were sensitive to the rate of rotation about the Co-O bond (Walker, F. A.; Bowen, J. H. J. Am. Chem. Sec. 1985, 107, 7632). We have now extended these studies to four metal-substituted hybrid hemoglobins in an attempt to determine whether EPR spectroscopy is sensitive to differences in the mobility of dioxygen in the alpha and beta subunits of the T and R quaternary states. For purposes of this study, [alpha(2)(CoO2)beta(2)(FeO2)] and [alpha(2)(FeO2)beta(2)(CoO2)] were used as R-state models and [alpha(2)(CoO2)beta(2)(Zn)] and [alpha(2)(Zn)beta(2)(CoO2)] were used as T-state models. EPR spectra were recorded for samples of each of the above hybrids, equilibrated with 1 atm of O-2 gas, as a function of temperature. The ''progress toward averaging'' of the EPR signals of the Co-O-2-containing subunits was measured as the difference in field positions, Delta H, for the midpoint of the low- and high-field extrema of the derivative EPR spectra. A plot of Delta H vs temperature for each hybrid shows that the [alpha(2)(Zn)beta(2)(CoO2)] hybrid is unique in averaging more slowly than the other three (all of which behave similarly), indicating more restricted rotation of dioxygen in T-state beta-chain pockets than in the heme distal O-2-binding pockets of any other form. This finding is consistent with X-ray crystallographic data which show that valine Ell on the distal side of the T-state P-chain pocket partially blocks the dioxygen binding site (Perutz, M. F.; Fermi, G.; Luisi, B.; Shaanan, B.; Liddington, R. C. Ace. Chem. Res. 1987, 20, 309). Simulation of EPR spectra as a function of jump time provides semiquantitative estimates of the rate of dioxygen rotation in these mixed-metal hemoglobin-dioxygen samples; these rates are in the 1 x 10(8) s(-1) range for three of the hybrids at 35-37 degrees C, and about one-third that value for T-state beta(CoO2) centers. These results provide new insight into the highly dynamic nature of dioxygen bound to the metal centers of hemoglobin at physiological temperatures.