Protein conformational relaxation and ligand migration in myoglobin: a nanosecond to millisecond molecular movie from time-resolved Laue X-ray diffraction.

A time-resolved Laue X-ray diffraction technique has been used to explore protein relaxation and ligand migration at room temperature following photolysis of a single crystal of carbon monoxymyoglobin. The CO ligand is photodissociated by a 7.5 ns laser pulse, and the subsequent structural changes are probed by 150 ps or 1 micros X-ray pulses at 14 laser/X-ray delay times, ranging from 1 ns to 1.9 ms. Very fast heme and protein relaxation involving the E and F helices is evident from the data at a 1 ns time delay. The photodissociated CO molecules are detected at two locations: at a distal pocket docking site and at the Xe 1 binding site in the proximal pocket. The population by CO of the primary, distal site peaks at a 1 ns time delay and decays to half the peak value in 70 ns. The secondary, proximal docking site reaches its highest occupancy of 20% at approximately 100 ns and has a half-life of approximately 10 micros. At approximately 100 ns, all CO molecules are accounted for within the protein: in one of these two docking sites or bound to the heme. Thereafter, the CO molecules migrate to the solvent from which they rebind to deoxymyoglobin in a bimolecular process with a second-order rate coefficient of 4.5 x 10(5) M(-1) s(-1). Our results also demonstrate that structural changes as small as 0.2 A and populations of CO docking sites of 10% can be detected by time-resolved X-ray diffraction.