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) por phyrinate(nitrogen base)(dioxygen) complexes in fluid solution were se nsitive to the rate of rotation about the Co-O bond (Walker, F. A.; Bo wen, J. H. J. Am. Chem. Sec. 1985, 107, 7632). We have now extended th ese 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 u sed as T-state models. EPR spectra were recorded for samples of each o f 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 fi eld positions, Delta H, for the midpoint of the low- and high-field ex trema 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 u nique in averaging more slowly than the other three (all of which beha ve similarly), indicating more restricted rotation of dioxygen in T-st ate 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-c hain 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 p rovides 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, an d about one-third that value for T-state beta(CoO2) centers. These res ults provide new insight into the highly dynamic nature of dioxygen bo und to the metal centers of hemoglobin at physiological temperatures.