This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Nitrite reductase (NiR) is a copper-containing enzyme, which carries out the first committed step of bacterial dissimilatory denitrification, the reduction of nitrite to nitric oxide. Nitrite reductase from the bacterium Alcaligenes faecalis serves as a model system for determination of high-resolution structures, which provide insight into this vital enzyme in biology. Data collected at SSRL resulted in a high-resolution structure of a Cu-nitrosyl, which showed an unexpected and unprecedented side-on NO coordination to the metal. Structures of NiR complexed with substrate and product have helped us revise the catalytic mechanism of nitrite reduction and have provided insights into the world of nitric oxide metal interactions in a biological system. The current structure of Cu(II)-NO- was achieved by reducing the crystals first and exposing them to NO. Since Cu(I)-NO+ is the proposed intermediate in the mechanism, a high-resolution structure of an oxidized enzyme interacting with NO is needed for supporting our current interpretations of catalysis. We are fortunate to have a biological system, which can serve as a model for copper NO chemistry and is also a major enzyme in denitrification. This system will be used to determine a high-resolution structure of the reaction intermediate, study pH dependence of NO binding and better understand the catalytic potential of NiR.
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