The molecular mechanism of the temperature enhancement of proton magnetic relaxation rates in methaemoprotein solutions

The mechanism of water exchange between the haem-pocket and bulk solvent in aqueous methaemopiotein solutions was firmly substantiated by using the aliphatic protons of certain lower alcohols in an otherwise deuterated solution for measuring the incremental relaxation rates resulting from their magnetic interaction with the haem-iron. The fast-exchange condition was established for solutions of horse fluorometmyoglobin, human A fluoromethaemoglobin and Chironomus thummi aquomethaemoglobin. The distances between the exchangeable protons and the haem-iron obtained from these PMR measurements concur with the presence of the fluoride ion, while for Chironomus aquomethaemoglobin this distance is also much larger than that resulting from the location of the 6th site Water molecule. The latter finding is the first clear-cut evidence that the exchanging protons belong to the next neighbour water molecule, a previously advanced hypothesis. The exchanging water molecule may thus serve as a natural probe for comparing the haem-pocket conformational state(s) under different conditions or in various haemoproteins.