Chemistry & Biochemistry
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The Department of Chemistry and Biochemistry offers research-oriented programs culminating in the Doctor of Philosophy degree. The faculty in the department have expertise over a broad range of specialty areas including synthesis, structure, spectroscopy, and mechanism. In each of these fields, the strength of the department has been recognized at the international level.
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Item The catalytic mechanism of electron bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD(2018-12) Schut, Gerrit J.; Mohamed-Raseek, Nishya; Tokmina-Lukaszewska, Monika; Mulder, David W.; Nguyen, Diep M. N.; Lipscomb, Gina L.; Hoben, John P.; Patterson, Angela; Lubner, Carolyn E.; King, Paul W.; Peters, John W.; Bothner, Brian; Miller, Anne-Frances; Adams, Michael W. W.Electron bifurcation plays a key role in anaerobic energy metabolism but it is a relatively new discovery and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon Pyrobaculum aerophilum The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using non-denaturing MS, cross-linking and homology modeling in which EtfA, B, and C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters. On the basis of analyses using transient absorption, EPR and optical titrations with NADH or inorganic reductants with and without NAD+, we propose a catalytic cycle involving formation of an intermediary NAD+-bound complex. A charge transfer signal revealed an intriguing interplay of flavin semiquinones and a protein conformational change that gated electron transfer between the low- and high-potential pathways. We found that despite a common bifurcating flavin site, the proposed EtfABCX catalytic cycle is distinct from that of the genetically-unrelated bifurcating NADH-dependent ferredoxin NADP+ oxidoreductase (NfnI). The two enzymes particularly differed in the role of NAD+, the resting and bifurcating-ready states of the enzymes, how electron flow is gated, and in the two two-electron cycles constituting the overall four-electron reaction. We conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETF\'s and can be applied to the large bifurcating ETF family.Item Mechanism of N2 Reduction Catalyzed by Fe-Nitrogenase Involves Reductive Elimination of H2(2018-02) Harris, Derek F.; Lukoyanov, Dmitriy A.; Shaw, Sudipta; Compton, Phil; Tokmina-Lukaszewska, Monika; Bothner, Brian; Kelleher, Neil; Dean, Dennis R.; Hoffman, Brian M.; Seefeldt, Lance C.Of the three forms of nitrogenase (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase), Fe-nitrogenase has the poorest ratio of N2 reduction relative to H2 evolution. Recent work on the Mo-nitrogenase has revealed that reductive elimination of two bridging Fe-H-Fe hydrides on the active site FeMo-cofactor to yield H2 is a key feature in the N2 reduction mechanism. The N2 reduction mechanism for the Fe-nitrogenase active site FeFe-cofactor was unknown. Here, we have purified both component proteins of the Fe-nitrogenase system, the electron-delivery Fe protein (AnfH) plus the catalytic FeFe protein (AnfDGK), and established its mechanism of N2 reduction. Inductively coupled plasma optical emission spectroscopy and mass spectrometry show that the FeFe protein component does not contain significant amounts of Mo or V, thus ruling out a requirement of these metals for N2 reduction. The fully functioning Fe-nitrogenase system was found to have specific activities for N2 reduction (1 atm) of 181 ± 5 nmol NH3 min-1 mg-1 FeFe protein, for proton reduction (in the absence of N2) of 1085 ± 41 nmol H2 min-1 mg-1 FeFe protein, and for acetylene reduction (0.3 atm) of 306 ± 3 nmol C2H4 min-1 mg-1 FeFe protein. Under turnover conditions, N2 reduction is inhibited by H2 and the enzyme catalyzes the formation of HD when presented with N2 and D2. These observations are explained by the accumulation of four reducing equivalents as two metal-bound hydrides and two protons at the FeFe-cofactor, with activation for N2 reduction occurring by reductive elimination of H2.Item Distinct properties underlie flavin-based electron bifurcation in a novel electron transfer flavoprotein FixAB from Rhodopseudomonas palustris(2018-02) Duan, H. D.; Lubner, Carolyn E.; Tokmina-Lukaszewska, Monika; Gauss, George H.; Bothner, Brian; King, Paul W.; Peters, John W.; Miller, A. F.A newly recognized third fundamental mechanism of energy conservation in biology, electron bifurcation, uses free energy from exergonic redox reactions to drive endergonic redox reactions. Flavin-based electron bifurcation furnishes low-potential electrons to demanding chemical reactions, such as reduction of dinitrogen to ammonia. We employed the heterodimeric flavoenzyme FixAB from the diazotrophic bacterium Rhodopseudomonas palustris to elucidate unique properties that underpin flavin-based electron bifurcation. FixAB is distinguished from canonical electron transfer flavoproteins (ETFs) by a second FAD that replaces the AMP of canonical ETF. We exploited near-UV–visible CD spectroscopy to resolve signals from the different flavin sites in FixAB and to interrogate the putative bifurcating FAD. CD aided in assigning the measured reduction midpoint potentials (E° values) to individual flavins, and the E° values tested the accepted model regarding the redox properties required for bifurcation. We found that the higher-E° flavin displays sequential one-electron (1-e−) reductions to anionic semiquinone and then to hydroquinone, consistent with the reactivity seen in canonical ETFs. In contrast, the lower-E° flavin displayed a single two-electron (2-e−) reduction without detectable accumulation of semiquinone, consistent with unstable semiquinone states, as required for bifurcation. This is the first demonstration that a FixAB protein possesses the thermodynamic prerequisites for bifurcating activity, and the separation of distinct optical signatures for the two flavins lays a foundation for mechanistic studies to learn how electron flow can be directed in a protein environment. We propose that a novel optical signal observed at long wavelength may reflect electron delocalization between the two flavins.Item H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle(2018-01) Berry, Luke; Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R.; Nguyen, Diep M. N.; Schut, Gerrit J.; Adams, Michael W. W.; Peters, John W.; Boyd, Eric S.; Bothner, BrianRecent investigations into ferredoxin-dependent transhydrogenases, a class of enzymes responsible for electron transport, have highlighted the biological importance of flavin-based electron bifurcation (FBEB). FBEB generates biomolecules with very low reduction potential by coupling the oxidation of an electron donor with intermediate potential to the reduction of high and low potential molecules. Bifurcating systems can generate biomolecules with very low reduction potentials, such as reduced ferredoxin (Fd), from species such as NADPH. Metabolic systems that use bifurcation are more efficient and confer a competitive advantage for the organisms that harbor them. Structural models are now available for two NADH-dependent ferredoxin-NADP(+) oxidoreductase (Nfn) complexes. These models, together with spectroscopic studies, have provided considerable insight into the catalytic process of FBEB. However, much about the mechanism and regulation of these multi-subunit proteins remains unclear. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and statistical coupling analysis (SCA), we identified specific pathways of communication within the model FBEB system, Nfn from Pyrococus furiosus, under conditions at each step of the catalytic cycle. HDX-MS revealed evidence for allosteric coupling across protein subunits upon nucleotide and ferredoxin binding. SCA uncovered a network of co-evolving residues that can provide connectivity across the complex. Together, the HDX-MS and SCA data show that protein allostery occurs across the ensemble of iron-sulfur cofactors and ligand binding sites using specific pathways that connect domains allowing them to function as dynamically coordinated units.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 Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation(2016-10) Kuznedelov, Konstantin; Mekler, Vladimir; Lemak, Sofia; Tokmina-Lukaszewska, Monika; Datsenko, Kirill A; Jain, Ishita; Savitskaya, Ekaterina; Mallon, John; Shmakov, Sergey; Bothner, Brian; Bailey, Scott; Yakunin, Alexander F; Severinov, KonstantinThe Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo Shortened crRNAs assemble into altered-stoichiometry Cascade effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multisubunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.