Light-induced relaxation of photolyzed carbonmonoxy myoglobin: a temperature-dependent x-ray absorption near-edge structure (XANES) study.

X-ray absorption near-edge structure (XANES) spectra at the Fe K-edge have been measured and compared on solution samples of horse carbonmonoxy-myoglobin and its photoproducts, prepared by two different photolysis protocols: 1), extended illumination at low temperature (15 K) by white light; and 2), slow-cool from 140 to 10 K at a rate of 0.5 K/min while illuminating the sample with a 532-nm continuous-wave laser source. CO recombination has been followed while increasing the temperature at a rate of 1.2 K/min. After extended illumination at 15 K, a single process is observed, corresponding to CO recombination from a completely photolyzed species with CO bound to the primary docking site (formally B-state, in agreement with previous x-ray diffraction studies). The temperature peak for this single process is approximately 50 K. Using slow-cool illumination, data show a two-state recombination curve, the two temperature peaks being roughly assigned to 50 K and 110 K. These results are in good agreement with previous FTIR studies using temperature-derivative spectroscopy. The XANES spectroscopic markers probe structural differences between the photoproduct induced by extended illumination at 15 K and the photoproduct induced by slow-cool illumination. These differences in the XANES data have been interpreted as due to light-induced Fe-heme relaxation that does not involve CO migration from the B-state. A quantitative description of the unrelaxed and relaxed B-states, including the measurements of the Fe-N(p), Fe-N(His), and Fe-CO distances, and the out-of-plane Fe displacement, has been obtained via a procedure (MXAN) recently developed by us. This work shows that XANES, being able to extract both kinetic and structural parameters in a single experiment, is a powerful tool for structural dynamic studies of proteins.