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Light-Induced Relaxation of Photolyzed Carbonmonoxy Myoglobin: A Temperature-Dependent X-Ray Absorption Near-Edge Structure (XANES) Study

A. Arcovito ^*, D. C. Lamb   , G. U. Nienhaus  , J. L. Hazemann ^¶, M. Benfatto ^|| and S. Della Longa ^|| **

^* Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm, Sweden; Department of Physical Chemistry and Center for Nanoscience, Ludwig-Maximilian-University Munich, Munich, Germany; Department of Biophysics, University of Ulm, Ulm, Germany; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois; ^¶ Laboratoire de Crystallographie, Centre National de la Recherche Scientifique, Grenoble, France; ^|| Laboratori Nazionali di Frascati (INFN), Frascati, Italy; and ^** Department of Experimental Medicine, University of L'Aquila, L'Aquila, Italy

Correspondence: Address reprint requests to S. Della Longa, Dip. Medicina Sperimentale, Universita' dell'Aquila, Via Vetoio, 67100 loc. Coppito, L'Aquila, Italy. Tel.: 39-0862-433568; Fax: 39-862-433523; E-mail: dlonga{at}caspur.it.

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 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.