Intermediate structure of normal human haemoglobin: methaemoglobin in the deoxy quaternary conformation.

Abstract This paper describes a new method of producing a crystalline intermediate between the unligated and ligated states of haemoglobin, suitable for X-ray analysis, by the use of a lattice strengthening reagent. Acrylamide is polymerized in the liquid of crystallization after the crystal has grown, forming a stiff supporting gel between the haemoglobin molecules, but not covalent bonds with them. The structure of human haemoglobin A crystallized in the deoxy quaternary structure (T-state † ) and then oxidized by air after lattice strengthening (tertiary structure made met, or r-state) was determined to 3.5 A resolution by the difference Fourier technique. Marked changes in tertiary structure in the region of the haem pockets and the contacts between the subunits (α 1 β 2 ) are observed. The iron is seen to move towards the plane of the porphyrin, causing a change of tilt of the haem. This appears to act as a lever setting in train stereochemical changes that loosen several hydrogen bonds within and between subunits, on which the stability of the tertiary and quaternary deoxy structures depend. The liganding water molecule itself causes a slight opening of the haem pocket in the α subunit, and a substantial one in the β subunit. The structural changes seen here in going from the tertiary deoxy to the aquomet state within the quaternary T-structure are similar, but opposite, to those seen earlier in going from aquomet to deoxy in the quaternary R-structure of BME-haemoglobin. Changes in tertiary structure associated with addition of ligand to the T-structure or the removal of ligand from the R-structure are thus seen to be complementary. Electron density maps show the α haems to undergo autoxidation more readily than the β haems, just as the β haems were reduced more easily than the α haems in BME-haemoglobin.