An X-ray study of horse methaemoglobin.

Abstract

The paper describes a detailed study of horse methaemoglobin by single crystal X-ray diffraction methods. The results give information on the arrangement of the molecules in the crystal, their shape and dimensions, and certain features of their internal structure. Horse methaemoglobin crystallizes in the monoclinic space group C 2 with two molecules of weight 66, 700 per unit cell. In addition, the wet crystals contain liquid of crystallization which fills 52.4% of the unit cell volume. Deliberate variations in the amount and com­position of the liquid of crystallization, and the study of the effects of such variations on the X-ray diffraction pattern, form the basis of the entire analysis. The composition of the liquid of crystallization can be varied by allowing heavy ions to diffuse into the crystals. This increases the scattering contribution of the liquid relative to that of the protein molecules and renders it possible to distinguish the one from the other. The method is analogous to that of isomorphous replacement commonly used in X-ray analysis. It yielded valuable information on the shape and character of the haemoglobin molecules and also led to the determination of the phase angles of certain reflexions. The amount of liquid of crystallization was varied by swelling and shrinkage of the crystals. This involves stepwise, reversible transitions between different well-defined lattices, each being stable in a particular environment of the crystal. The lattice changes were utilized in two different ways: the first involved comparison of Patters on projections at different stages of swelling and shrinkage, and the second an attempt to trace the molecular scattering curve as a function of the diffraction angle. The results of the analysis can be summarized as follows. The methaemoglobin molecules resemble cylinders of an average height of 34 A and a diameter of 57 A. In the crystal these cylinders form close-packed layers which alternate with layers of liquid of crystallization. The layers of haemoglobin molecules themselves do not swell or shrink, either in thickness or in area, except on complete drying, and lattice changes merely involve a shearing of the haemoglobin layers relative to each other, combined with changes in the thickness of the liquid layer. Thus the molecules do not seem to be penetrated by the liquid of crystallization, and their structure is unaffected by swelling and shrinkage of the crystal. Space-group symmetry requires that each molecule consists of two chemically and struc­turally identical halves. Evidence concerning the internal structure of the molecules comes both from two-dimensional Patterson projections and one-dimensional Fourier projections. The former indicate that interatomic vectors of 9 to 11 A occur frequently in many directions, and the latter show four prominent concentrations of scattering matter just under 9 A apart along a line normal to the layers of haemoglobin molecules. No structural interpretation of these features is as yet attempted. The liquid of crystallization consists of two distinct components: water ‘bound’ to the protein and not available as solvent to diffusing ions, and ‘free’ water in dynamic equilibrium with the suspension medium. An estimate of the ‘frictional ratio’ based on the molecular shape and hydration found in this analysis is in good agreement with the frictional ratio calculated from the sedimentation constant.