Browsing by Author "Adams, Michael W. W."

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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.
Defining Electron Bifurcation in the Electron Transferring Flavoprotein Family
(2017-11) Garcia Costas, Amaya M.; Poudel, Saroj; Miller, Anne-Frances; Schut, Gerrit J.; Ledbetter, Rhesa N.; Fixen, Kathryn R.; Seefeldt, Lance C.; Adams, Michael W. W.; Harwood, Caroline S.; Boyd, Eric S.; Peters, John W.
Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low and high potential electrons. It is the third recognized form of energy conservation in biology and has recently been described in select electron transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via ETF quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer as well as a non-redox active adenosine monophosphate (AMP). However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatics and structural analyses. Etfs were identified in diverse archaea and bacteria, and these clustered into five distinct well-supported groups based on amino acid sequences. Gene neighborhood analyses indicate that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting distinct and conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH and FAD-binding regions of bifurcating Etfs. Collectively, a new classification scheme is presented for Etf proteins that demarcates putative bifurcating vs. non-bifurcating members and suggests that Etf mediated bifurcation is associated with surprisingly diverse enzymes.IMPORTANCE Electron bifurcation has recently been recognized as an electron transfer mechanism used by microorganisms to maximize energy conservation. Bifurcating enzymes couple thermodynamically unfavorable reactions with thermodynamically favorable reactions in an overall spontaneous process. Here we show that the electron transferring flavoprotein (Etf) enzyme family exhibits far greater diversity than previously recognized and we provide a phylogenetic analysis that clearly delineates bifurcating and non-bifurcating members of this family. Structural modeling of proteins within these groups reveals key differences between the bifurcating and non-bifurcating Etfs.
[FeFe]- and [NiFe]-hydrogenase diversity, mechanism, and maturation
(2014-11) Peters, John W.; Schut, Gerrit J.; Boyd, Eric S.; Mulder, David W.; Shepard, Eric M.; Broderick, Joan B.; King, Paul W.; Adams, Michael W. W.
The [FeFe]- and [NiFe]-hydrogenases catalyze the formal interconversion between hydrogen and protons and electrons, possess characteristic non-protein ligands at their catalytic sites and thus share common mechanistic features. Despite the similarities between these two types of hydrogenases, they clearly have distinct evolutionary origins and likely emerged from different selective pressures. [FeFe]-hydrogenases are widely distributed in fermentative anaerobic microorganisms and likely evolved under selective pressure to couple hydrogen production to the recycling of electron carriers that accumulate during anaerobic metabolism. In contrast, many [NiFe]-hydrogenases catalyze hydrogen oxidation as part of energy metabolism and were likely key enzymes in early life and arguably represent the predecessors of modern respiratory metabolism. Although the reversible combination of protons and electrons to generate hydrogen gas is the simplest of chemical reactions, the [FeFe]- and [NiFe]-hydrogenases have distinct mechanisms and differ in the fundamental chemistry associated with proton transfer and control of electron flow that also help to define catalytic bias. A unifying feature of these enzymes is that hydrogen activation itself has been restricted to one solution involving diatomic ligands (carbon monoxide and cyanide) bound to an Fe ion. On the other hand, and quite remarkably, the biosynthetic mechanisms to produce these ligands are exclusive to each type of enzyme. Furthermore, these mechanisms represent two independent solutions to the formation of complex bioinorganic active sites for catalyzing the simplest of chemical reactions, reversible hydrogen oxidation. As such, the [FeFe]- and [NiFe]-hydrogenases are arguably the most profound case of convergent evolution. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.
Genomic Features and Pervasive Negative Selection in Rhodanobacter Strains Isolated from Nitrate and Heavy Metal Contaminated Aquifer
(American Society for Microbiology, 2022-02) Peng, Mu; Wang, Dongyu; Lui, Lauren M.; Nielsen, Torben; Tian, Renmao; Kempher, Megan L.; Tao, Xuanyu; Pan, Chongle; Chakraborty, Romy; Deutschbauer, Adam M.; Thorgersen, Michael P.; Adams, Michael W. W.; Fields, Matthew W.; Hazen, Terry C.; Arkin, Adam P.; Zhou, Aifen; Zhou, Jizhong
Despite the dominance of Rhodanobacter species in the subsurface of the contaminated Oak Ridge Reservation (ORR) site, very little is known about the mechanisms underlying their adaptions to the various stressors present at ORR. Recently, multiple Rhodanobacter strains have been isolated from the ORR groundwater samples from several wells with varying geochemical properties.
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, Brian
Recent 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.
Microbial Functional Gene Diversity Predicts Groundwater Contamination and Ecosystem Functioning
(2018-02) He, Zhili; Zhang, Ping; Wu, Linwei; Rocha, Andrea M.; Tu, Qichao; Shi, Zhou; Wu, Bo; Qin, Yujia; Wang, Jianjun; Yan, Qingyun; Curtis, Daniel; Ning, Daliang; Van Nostrand, Joy D.; Wu, Liyou; Yang, Yunfeng; Elias, Dwayne A.; Watson, David B.; Adams, Michael W. W.; Fields, Matthew W.; Alm, Eric J.; Hazen, Terry C.; Adams, Paul D.; Arkin, Adam P.; Zhou, Jizhong
Contamination from anthropogenic activities has significantly impacted Earth\'s biosphere. However, knowledge about how environmental contamination affects the biodiversity of groundwater microbiomes and ecosystem functioning remains very limited. Here, we used a comprehensive functional gene array to analyze groundwater microbiomes from 69 wells at the Oak Ridge Field Research Center (Oak Ridge, TN), representing a wide pH range and uranium, nitrate, and other contaminants. We hypothesized that the functional diversity of groundwater microbiomes would decrease as environmental contamination (e.g., uranium or nitrate) increased or at low or high pH, while some specific populations capable of utilizing or resistant to those contaminants would increase, and thus, such key microbial functional genes and/or populations could be used to predict groundwater contamination and ecosystem functioning. Our results indicated that functional richness/diversity decreased as uranium (but not nitrate) increased in groundwater. In addition, about 5.9% of specific key functional populations targeted by a comprehensive functional gene array (GeoChip 5) increased significantly (P < 0.05) as uranium or nitrate increased, and their changes could be used to successfully predict uranium and nitrate contamination and ecosystem functioning. This study indicates great potential for using microbial functional genes to predict environmental contamination and ecosystem functioning.IMPORTANCE Disentangling the relationships between biodiversity and ecosystem functioning is an important but poorly understood topic in ecology. Predicting ecosystem functioning on the basis of biodiversity is even more difficult, particularly with microbial biomarkers. As an exploratory effort, this study used key microbial functional genes as biomarkers to provide predictive understanding of environmental contamination and ecosystem functioning. The results indicated that the overall functional gene richness/diversity decreased as uranium increased in groundwater, while specific key microbial guilds increased significantly as uranium or nitrate increased. These key microbial functional genes could be used to successfully predict environmental contamination and ecosystem functioning. This study represents a significant advance in using functional gene markers to predict the spatial distribution of environmental contaminants and ecosystem functioning toward predictive microbial ecology, which is an ultimate goal of microbial ecology.
Smartphone Analytics: Mobilizing the Lab into the Cloud for Omic-Scale Analyses
(2016-08) Montenegro-Burke, Jose R.; Phommavongsay, Thiery; Aisporna, Aries E.; Huan, Tao; Rinehart, Duane; Forsberg, Erica M.; Poole, Farris L.; Thorgersen, Michael P.; Adams, Michael W. W.; Krantz, Gregory; Fields, Matthew W.; Northen, Trent R.; Robbins, Paul D.; Niedernhofer, Laura J.; Lairson, Luke L.; Benton, H. Paul; Siuzdak, Gary
Active data screening is an integral part of many scientific activities, and mobile technologies have greatly facilitated this process by minimizing the reliance on large hardware instrumentation. In order to meet with the increasingly growing field of metabolomics and heavy workload of data processing, we designed the first remote metabolomic data screening platform for mobile devices. Two mobile applications (apps), XCMS Mobile and METLIN Mobile, facilitate access to XCMS and METLIN, which are the most important components in the computer-based XCMS Online platforms. These mobile apps allow for the visualization and analysis of metabolic data throughout the entire analytical process. Specifically, XCMS Mobile and METLIN Mobile provide the capabilities for remote monitoring of data processing, real time notifications for the data processing, visualization and interactive analysis of processed data (e.g., cloud plots, principle component analysis, box-plots, extracted ion chromatograms, and hierarchical cluster analysis), and database searching for metabolite identification. These apps, available on Apple iOS and Google Android operating systems, allow for the migration of metabolomic research onto mobile devices for better accessibility beyond direct instrument operation. The utility of XCMS Mobile and METLIN Mobile functionalities was developed and is demonstrated here through the metabolomic LC-MS analyses of stem cells, colon cancer, aging, and bacterial metabolism.
Systems biology guided by XCMS Online metabolomics
(2017-04) Huan, Tao; Forsberg, Erica M.; Rinehart, Duane; Johnson, Caroline H.; Ivanisevic, Julijana; Benton, H. Paul; Fang, Mingliang; Aisporna, Aries E.; Hilmers, Brian; Poole, Farris L.; Thorgersen, Michael P.; Adams, Michael W. W.; Krantz, Gregory; Fields, Matthew W.; Robbins, Paul D.; Niedernhofer, Laura J.; Ideker, Trey; Majumder, Erica L.; Wall, Judy D.; Rattray, Nicholas J. W.; Goodacre, Royston; Lairson, Luke L.; Siuzdak, Gary
Two functionally distinct NADP(+)-dependent ferredoxin oxidoreductases maintain the primary redox balance of Pyrococcus furiosus
(2017-07) Nguyen, Diep M. N.; Schut, Gerrit J.; Zadvornyy, Oleg A.; Tokmina-Lukaszewska, Monika; Poudel, Saroj; Lipscomb, Gina L.; Adams, Leslie A.; Dinsmore, Jessica T.; Nixon, William J.; Boyd, Eric S.; Bothner, Brian; Peters, John W.; Adams, Michael W. W.
Electron bifurcation has recently gained acceptance as the third mechanism of energy conservation in which energy is conserved through the coupling of exergonic and endergonic reactions. A structure-based mechanism of bifurcation has been elucidated recently for the flavin-based enzyme NADH-dependent ferredoxin NADP+ oxidoreductase I (NfnI) from the hyperthermophillic archaeon Pyrococcus furiosus. NfnI is thought to be involved in maintaining the cellular redox balance, producing NADPH for biosynthesis by recycling the two other primary redox carriers, NADH and ferredoxin. The P. furiosus genome encodes an NfnI paralog termed NfnII, and the two are differentially expressed depending on the growth conditions. In this study, we show that deletion of the genes encoding either NfnI or NfnII affects the cellular concentrations of NAD(P)H and particularly NADPH. This results in a moderate to severe growth phenotype in deletion mutants, demonstrating a key role for each enzyme in maintaining redox homeostasis. Despite their similarity in primary sequence and cofactor content, crystallographic, kinetic, and mass spectrometry analyses reveal that there are fundamental structural differences between the two enzymes and NfnII does not catalyze the NfnI bifurcating reaction. Instead it exhibits non-bifurcating ferredoxin NADP oxidoreductase-type activity. NfnII is therefore proposed to be a bifunctional enzyme and to also catalyze a bifurcating reaction, although its third substrate, in addition to ferredoxin and NADP(H), is as yet unknown.
Unification of [FeFe]-hydrogenases into Three Structural and Functional Groups
(2016-09) Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R.; Refai, Mohammed Y.; Schut, Gerrit J.; King, Paul W.; Maness, Pin-Ching; Adams, Michael W. W.; Peters, John W.; Bothner, Brian; Boyd, Eric S.
Background: [FeFe]-hydrogenases (Hyd) are structurally diverse enzymes that catalyze the reversible oxidation of hydrogen (H2). Recent biochemical data demonstrate new functional roles for these enzymes, including those that function in electron bifurcation where an exergonic reaction is coupled with an endergonic reaction to drive the reversible oxidation/production of H2. Methods: To identify the structural determinants that underpin differences in enzyme functionality, a total of 714 homologous sequences of the catalytic subunit, HydA, were compiled. Bioinformatics approaches informed by biochemical data were then used to characterize differences in inferred quaternary structure, HydA active site protein environment, accessory iron-sulfur clusters in HydA, and regulatory proteins encoded in HydA gene neighborhoods. Results: HydA homologs were clustered into one of three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while those in G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3) subunits. Variation in the HydA active site and accessory iron-sulfur clusters did not vary by group type. Group-specific regulatory genes were identified in the gene neighborhoods of both G2 and G3 Hyd. Analyses of purified G2 and G3 enzymes by mass spectrometry strongly suggest that they are post-translationally modified by phosphorylation. Conclusions: These results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes, rather than by variation in HydA. General significance: This classification scheme provides a framework for future biochemical and mutagenesis studies to elucidate the functional role of Hyd enzymes.