Scholarship & Research
Permanent URI for this communityhttps://scholarworks.montana.edu/handle/1/1
Browse
4 results
Search Results
Item Unraveling the interactions of the physiological reductant flavodoxin with the different conformations of the Fe protein in the nitrogenase cycle(2017-08) Pence, Natasha; Tokmina-Lukaszewska, Monika; Yang, Zhi-Yong; Ledbetter, Rhesa N.; Seefeldt, Lance C.; Bothner, Brian; Peters, John W.Nitrogenase reduces dinitrogen (N2) to ammonia in biological nitrogen fixation. The nitrogenase Fe protein cycle involves a transient association between the reduced, MgATP-bound Fe protein and the MoFe protein and includes electron transfer, ATP hydrolysis, release of Pi , and dissociation of the oxidized, MgADP-bound Fe protein from the MoFe protein. The cycle is completed by reduction of oxidized Fe protein and nucleotide exchange. Recently, a kinetic study of the nitrogenase Fe protein cycle involving the physiological reductant flavodoxin reported a major revision of the rate-limiting step from MoFe protein and Fe protein dissociation, to release of Pi . Since the Fe protein cannot interact with flavodoxin and the MoFe protein simultaneously, knowledge of the interactions between flavodoxin and the different nucleotide states of the Fe protein is critically important for understanding the Fe protein cycle. Here, we used time-resolved limited proteolysis and chemical cross-linking to examine nucleotide-induced structural changes in the Fe protein and their effects on interactions with flavodoxin. Differences in proteolytic cleavage patterns and chemical cross-linking patterns were consistent with known nucleotide-induced structural differences in the Fe protein and indicated that MgATP-bound Fe protein resembles the structure of the Fe protein in the stabilized nitrogenase complex structures. Docking models and cross-linking patterns between the Fe protein and flavodoxin revealed that the MgADP-bound state of the Fe protein has the most complementary docking interface with flavodoxin compared with the MgATP-bound state. Together, these findings provide new insights into the control mechanisms in protein-protein interactions during the Fe protein cycle.Item Identification and characterization of a novel member of the radical AdoMet enzyme superfamily and implications for the biosynthesis of the Hmd hydrogenase active site cofactor(2009-11) McGlynn, Shawn E.; Boyd, Eric S.; Shepard, Eric M.; Lange, Rachel K.; Gerlach, Robin; Broderick, Joan B.; Peters, John W.The genetic context, phylogeny, and biochemistry of a gene flanking the H2-forming methylene-H4-methanopterin dehydrogenase gene (hmdA), here designated hmdB, indicate that it is a new member of the radical S-adenosylmethionine enzyme superfamily. In contrast to the characteristic CX3CX2C or CX2CX4C motif defining this family, HmdB contains a unique CX5CX2C motif.Item Diversity, abundance, and potential activity of nitrifying and nitrate-reducing microbial assemblages in a subglacial ecosystem(2011-05) Boyd, Eric S.; Lange, Rachel K.; Mitchell, Andrew C.; Havig, Jeff R.; Lafreniere, M. J.; Shock, Everett L.; Peters, John W.; Skidmore, Mark L.Subglacial sediments sampled from beneath Robertson Glacier (RG), Alberta, Canada, were shown to harbor diverse assemblages of potential nitrifiers, nitrate reducers, and diazotrophs, as assessed by amoA, narG, and nifH gene biomarker diversity. Although archaeal amoA genes were detected, they were less abundant and less diverse than bacterial amoA, suggesting that bacteria are the predominant nitrifiers in RG sediments. Maximum nitrification and nitrate reduction rates in microcosms incubated at 4°C were 280 and 18.5 nmol of N per g of dry weight sediment per day, respectively, indicating the potential for these processes to occur in situ. Geochemical analyses of subglacial sediment pore waters and bulk subglacial meltwaters revealed low concentrations of inorganic and organic nitrogen compounds. These data, when coupled with a C/N atomic ratio of dissolved organic matter in subglacial pore waters of ∼210, indicate that the sediment communities are N limited. This may reflect the combined biological activities of organic N mineralization, nitrification, and nitrate reduction. Despite evidence of N limitation and the detection of nifH, we were unable to detect biological nitrogen fixation activity in subglacial sediments. Collectively, the results presented here suggest a role for nitrification and nitrate reduction in sustaining microbial life in subglacial environments. Considering that ice currently covers 11% of the terrestrial landmass and has covered significantly greater portions of Earth at times in the past, the demonstration of nitrification and nitrate reduction in subglacial environments furthers our understanding of the potential for these environments to contribute to global biogeochemical cycles on glacial-interglacial timescales.Item Photo-induced H2 production by [NiFe]-hydrogenase from T. roseopersicina covalently linked to a Ru(II) photosensitizer(2012-01) Zadvornyy, Oleg A.; Lucon, Janice E.; Gerlach, Robin; Zorin, Nikolay A.; Douglas, Trevor; Elgren, T. E.; Peters, John W.The potential of hydrogen as a clean renewable fuel source and the finite reserves of platinum metal to be utilized in hydrogen production catalysts have provided the motivation for the development of non-noble metal-based solutions for catalytic hydrogen production. There are a number of microorganisms that possess highly efficient hydrogen production catalysts, termed hydrogenases, that generate hydrogen under certain metabolic conditions. Although hydrogenases occur in photosynthetic microorganisms, the oxygen sensitivity of these enzymes represents a significant barrier in directly coupling hydrogen production to oxygenic photosynthesis. To overcome this barrier, there has been considerable interest in identifying or engineering oxygen tolerant hydrogenases or generating mimetic systems that do not rely on oxygen producing photocatalysts. In this work, we demonstrate photo-induced hydrogen production from a stable [NiFe]-hydrogenase coupled to a [Ru(2,2'-bipyridine)2(5-amino1,10 phenanthroline)]2+ photocatalyst. When the Ru(II) complex is covalently attached to the hydrogenase, photocatalytic hydrogen production occurs more efficiently in the presence of a redox mediator than if the Ru(II) complex is simply present in solution. Furthermore, sustained hydrogen production occurs even in the presence of oxygen by presumably creating a local anoxic environment through the reduction of oxygen similar to what is proposed for oxygen tolerant hydrogenases. These results provide a strong proof of concept for engineering photocatalytic hydrogen production in the presence of oxygen using biohybrid mimetic systems.