Structural distribution and rotational disorder in myoglobin crystals.

Spectroscopic measurements provide not only energy level differences but, with appropriate experimental conditions and analysis, also widths of levels. Widths can often be shown to be due primarily to distribution in structure at the spectroscopically active center, such as at the high-spin ferric ion in myoglobin crystals discussed below. The g-tensor of high-spin ferric ion in heme is highly anisotropic. This property has been used in several laboratories to estimate the disorder in heme normal directions in crystals. For aand fl-chains of horse hemoglobin, 37% of the normals lie outside of a cone of half-angle flD = 40 (Hampton and Brill, 1979); in sperm whale myoglobin, fD is half this value (Fiamingo, 1980). While rotational disorder corresponding to fD = 20 is the dominant source of line broadening for many orientations of magnetic field, H, it contributes less than a gauss to observed peak-to-trough linewidth when H is parallel to the heme plane. Dipolar interactions are somewhat more effective in producing broadening in the heme plane, but they and misorientation together are of minor influence in comparison with structural distribution. At 37.5 GHz, Helcke et al. (1968) observed 250% variation in linewidth of electron resonances from the heme plane of aquo myoglobin crystals. In conjunction with measurements of orientation and temperature dependence of power saturation and spin-lattice relaxation time, we have measured the orientation dependence of linewidth at 9.27 GHz (Fiamingo, 1980). These data, together with simulations based upon known hyperfine coupling constants, provide hyperfine component linewidths, the squares of which show a heme plane angular dependence L + M cos 24 + N cos(40-fl) with L, M, and N = 159, -22, and -72 G2, respectively, and 6' = 1220 (Fig. 1). Calvo and Bemski (1976) proposed independently fluctuating principal directions and values of the zero-field splitting tensor to