Redox-induced structural dynamics of Fe-heme-ligand in myoglobin by X-ray absorption spectroscopy

The Fe(III) => Fe(II) reduction of the heme iron in aquomet-myoglobin, induced by x-rays at cryogenics temperatures, produces a thermally trapped nonequilibrium state in which a water molecule is still bound to the iron. Water dissociates at T > 160 K, when the protein can relax toward its new equilibrium, deoxy form. Synchrotron radiation x-ray absorption spectroscopy provides information on both the redox state and the Fe-heme structure. Owing to the development of a novel method to analyze the low-energy region of x-ray absorption spectroscopy, we obtain structural pictures of this photoinducible, irreversible process, with 0.02–0.06 Å accuracy, on the protein in solution as well as in crystal. After photo-reduction, the iron-proximal histidine bond is shortened by 0.15 Å , a reinforcement that should destabilize the iron in-plane position favouring water dissociation. Moreover, we are able to get the distance of the water molecule even after dissociation from the iron, with a 0.16-Å statistical error.