This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Heme oxygenase (HO) catalyzes physiological degradation of heme using O2 and reducing equivalents to produce biliverdin IXalpha (BV) ferrous iron and CO (Tenhunen R. et al. (1968) Proc. Natl. Acad. Sci. USA 61 748-755). The HO reaction proceeds without product inhibition by the CO generated in the second step of the HO reaction although CO is known to be a potent inhibitor of HO and other heme proteins. In fact exogenous CO inhibits the overall HO reaction especially its third step. Therefore HO must have a mechanism by which it escapes inhibition by endogenously produced CO. In order to track how endogenous CO is released from the reaction site we previously collected two X-ray diffraction data sets from the CO-bound form of one crystal of the hemeHO complex. One data set was collected in the dark and the other under illumination by He-Ne CW laser at 35 K. The light minus dark difference Fourier map showed that CO was partially photodissociated from the heme iron and dissociated CO was trapped in a hydrophobic cavity adjacent to the heme pocket (Sugishima M. et al. (2004) J. Mol. Biol. 341 7-13). In contrast to results on globins we could not detect any protein motion upon photodissociation because the extent of photodissociation was low and the cryogenic conditions severely restrict protein motion. To overcome these issues I propose time-resolved Laue experiments at room temperature and cryo-trapping experiments at higher temperatures where protein motion is less restricted. Using these approaches the motions of protein and heme upon CO photodissociation which mimic the endogenous CO dissociation process could be readily detectable. In addition I also propose to identify reaction intermediates during the HO reaction in the crystal without the necessity for chemical or cryotrapping. Previously we initiated the HO reaction in crystals by diffusing a reducing reagent into the initially oxidized form of heme-HO complex crystals. We successfully determined the crystal structure of one of the reaction intermediates the bound form of BV-iron chelate (Sugishima M. et al. (2003) J. Biol. Chem. 278 32352-8). Because a long time (~3hr) is required for the completion of the overall reaction several other intermediate states can be easily generated as the reaction proceeds. We did not analyze the intermediate state structures at that time because we expected that several reaction intermediate states were present in a complex mixture at all time points. However the algorithms and software for analysis of such mixtures have been developed (Schmidt M. et al. (2003) Biophys. J. 84 2112-29). Details of the intermediate structures the overall mechanism and the rate coefficients for interconversion of these intermediates during the HO reaction would be revealed by applying this method to the diffraction datasets obtained during the reaction. Depending on the time required to acquire a complete data set we could use either the Laue or monochromatic oscillation techniques. In plants red algae and cyanobacteria BV produced by HO is reduced to phytochromobilin and phycobilins by ferredoxin-dependent bilin reductases. PcyA one of the enzymes in this family catalyzes the reduction of BV to phycocyanobilin which is incorporated into light-harvesting and light-sensing complexes in red algae and cyanobacteria. Previously we determined the structure of PcyA in complex with BV (Hagiwara Y. et al. (2006) Proc. Natl. Acad. Sci. USA 103 27-32). We found an unexpected covalent link between PcyA and BV under room light illumination;this covalent link was not found under dark conditions. This photochemical reaction may occur in vivo because red algae and cyanobacteria are photosynthetic organisms. To characterize this covalent link I propose time-resolved and cryo-trapping X-ray data collection from PcyA crystals in the BV-bound form in the dark and under laser illumination.

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