To understand the structural basis for metalloprotein activity and conformational control at the atomic level, we have successfully integrated crystallographic studies for four proteins that undergo functionally-important structural and spectroscopic changes. We chose the photoactive yellow protein (PYP) together with the Root-effect hemoglobin (Hb), active hemoprotein subunit of sulfite reductase (SiRHP), and Cu,Zn superoxide dismutase (SOD) metalloproteins for their biological importance. The integrated study of these 4 proteins aims to identify common and variable elements in the mechanisms of functionally- important conformational change in metalloproteins and other chromatically active proteins. The chromophore spectra are probes of each protein's conformational state in the crystals and allow the atomic structures to be complemented by detailed electronic information from spectroscopy. Crystals of PYP were grown that diffract to at least 1.3angstroms resolution, and a 2angstroms structure was determined. The crystals can be bleached by visible light, but are not bleached by x- rays. We will determine and analyze atomic structures of bleached, unbleached, and time-resolved intermediates of PYP, deduce the nature of photo-induced conformational change, and compare PYP structural changes to those in the three metalloproteins. The extreme effect of pH on the oxygen affinity of Root-effect Hbs allows fish to pump oxygen into the swim bladder against 100 atmospheres of oxygen pressure. We grew crystals for the R and T state conformation of Leiostomas xanthurus Root-effect Hb and refined and analyzed the R-state structure at 2.3angstroms resolution. We propose to further refine the R-state structure, determine the T-state structure, and establish the structural basis for the extreme pH dependence of O2 binding. We crystallized the 6-electron reducing enzyme SiRHP, collected 1.7angstroms resolution native data, solved heavy atom derivatives, and determined a partial model. We will complete SiRHP structure determination in oxidized, reduced and sulfite-bound conformations, determine important mutant structures, and elucidate the structural implications for the enzyme mechanism. This will provide the first structural data on a protein containing both a siroheme and an Fe4S4 cluster and will serve as a model for other multi-center multiple electron transfer enzymes, such as cytochrome oxidase, nitrogenase, and nitrite reductase. We solved atomic structures of bovine SOD (BSOD) in oxidized, reduced and inhibitor bound states. We obtained crystals and 2.1angstroms resolution data for Photobacter leiognathi SOD (PSOD), which shares little sequence homology with other SODs and represents the subclass of SOD from pathogens and symbionts. Wild-type and mutant PSOD structures will be determined and compared to the known SOD structures. Quantum calculations on BSOD orbitals in the different structural states will allow direct correlations of these structures to the enzyme's spectroscopy and mechanism. Taken together, this research on PYP, Hb, SiRHP, and SOD will contribute specifically to defining the atomic basis for their structural changes and activities, and more generally to identifying mechanisms of functionally-important conformational change fundamental to many systems of biological and medical interest.
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