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.