title: Light-induced relaxation of photolyzed carbonmonoxy myoglobin: a temperature-dependent x-ray absorption near-edge structure (XANES) study.
abstract: 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.
authors.affiliation: Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm, Sweden.
authors.forename: A
authors.initials: A
authors.lastname: Arcovito
authors.forename: D C
authors.initials: DC
authors.lastname: Lamb
authors.forename: G U
authors.initials: GU
authors.lastname: Nienhaus
authors.forename: J L
authors.initials: JL
authors.lastname: Hazemann
authors.forename: M
authors.initials: M
authors.lastname: Benfatto
authors.forename: S
authors.initials: S
authors.lastname: Della Longa
mesh_terms: D000818:Animals; D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry; D018360:Crystallography, X-Ray; D006418:Heme / Q000737:chemistry; D006736:Horses; D008027:Light; D008956:Models, Chemical; D008958:Models, Molecular; D009211:Myoglobin / Q000737:chemistry*; D010782:Photolysis; D011485:Protein Binding; D011487:Protein Conformation; D011995:Recombination, Genetic; D012984:Software; D013052:Spectrometry, X-Ray Emission / Q000379:methods*; D013053:Spectrophotometry / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D014961:X-Ray Diffraction; D014965:X-Rays
chemical_list: D009211:Myoglobin; C018013:carboxymyoglobin; D006418:Heme; D002248:Carbon Monoxide