i The Pennsylvania State University The Graduate School Eberly College of Science THE ELECTRON TRANSPORT OF ACETATE-GROWN METHANOSARCINA ACETIVORANS A Dissertation in Biochemistry, Microbiology, and Molecular Biology by Mingyu Wang 2010 Mingyu Wang Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2010 i The dissertation of Mingyu Wang was reviewed and approved* by the following: James G. Ferry Stanley Person Professor and Director, Center for Microbial Structural Biology Dissertation Advisor Chair of Committee Sarah E. Ades Associate Professor of Biochemistry and Molecular Biology Donald A. Bryant Ernest C. Pollard Professor of Biotechnology and Professor of Biochemistry and Molecular Biology Christopher H. House Associate Professor of Geosciences Ming Tien Professor of Biochemistry Scott B. Selleck Professor and Head, Department of Biochemistry and Molecular Biology *Signatures are on file in the Graduate School iii ABSTRACT The electron transport of marine acetate-utilizing methanogen Methanosarcina acetivorans was investigated, leading to the first identification and partial characterization of two novel ferredoxin: CoM-S-S-CoB electron transport chains. The study of an Rnf-dependent membrane-bound pathway of aceticlastic M. acetivorans that doesn’t reduce CO2 with H2 characterized members both unique to Rnf-dependent pathway and also in common with Ech-dependent ferredoxin: CoM-S-S-CoB electron transport chain in CO2 utilizing aceticlastic methanogens. These include the Rnf complex, cytochrome c, ferredoxin, Cdh, methanophenazine, heterodisulfide reductase and CoM-S-S-CoB. The purification and phylogenetic analysis of ferredoxin suggested an aceticlastic-specific ferredoxin clade among methanogens. This Rnf-dependent electron transport pathway, shared with non-CO2 utilizing Methanosarcina thermophila is analogous to Ech-dependent electron transport pathway in its location and terminal electron partners but differs from the later in that it lacks the use of hydrogenases and H2. A novel soluble ferredoxin: CoM-S-S-CoB pathway was identified in M. acetivorans by comparing soluble and membrane-bound ferredoxin: CoM-S-S-CoB oxidoreductase activities and was hypothesized to involve a HdrA: MvhD fusion protein, named Etp. Etp was heterologously overexpressed in Escherichia coli, purified, reconstituted and partially characterized for the first time, showing heavy iron-sulfur cluster content. The reduction of Etp is linked to the oxidation of ferredoxin mediated by unknown soluble factors. Finally, a model of energy conservation for M. acetivorans was constructed suggesting this soluble electron transport chain serves to bypass certain energy coupling sites and maximize energy conservation efficiency under substrate-limited scenarios. iv TABLE OF CONTENTS LIST OF FIGURES .............................................................................................. viii LIST OF EQUATIONS ........................................................................................ xii LIST OF TABLES ................................................................................................ xiv ACKNOWLEDGEMENTS .................................................................................. xv Chapter 1 Methanogens and methanogenesis .............................................................. 1 1.1 Methanogenesis and methanogens ................................................................. 1 1.1.1 Methanogenesis .................................................................................... 1 1.1.2 Ecology of methanogens ...................................................................... 2 1.1.3 Taxonomy and cell biology of methanogens ........................................ 4 1.2 Methanogenesis from three major biochemical pathways .............................. 6 1.2.1 Cofactors utilized in methanogenic pathways ...................................... 6 1.2.2 H2 + CO2 pathway ................................................................................ 9 1.2.3 Methylotrophic pathway ....................................................................... 25 1.2.4 Acetate pathway ................................................................................... 31 1.3 References ....................................................................................................... 36 Chapter 2 Electron transport and energy conservation in methanogens ...................... 69 2.1 Introduction ..................................................................................................... 69 2.2 Methanogenesis coupled ATP synthesis ........................................................ 69 2.3 Membrane-bound electron transport chains of methanogens ......................... 72 v 2.3.1 F420: CoM-S-S-CoB pathway ............................................................... 73 2.3.2 H2: CoM-S-S-CoB pathway ................................................................. 74 2.3.3 The acetate-specific ferredoxin: CoM-S-S-CoB electron transport chain ....................................................................................................... 76 2.4 Electron transport in obligate CO2-reducing methanogens ............................ 78 2.5 Enzymes involved in electron transport chains of methanogens .................... 80 2.5.1 F420 dehydrogenase ............................................................................... 80 2.5.2 Membrane bound F420 non-reducing hydrogenase ............................... 83 2.5.3 Ech hydrogenase ................................................................................... 85 2.5.4 Rnf complex ......................................................................................... 87 2.6 References ....................................................................................................... 90 Chapter 3 The membrane-bound electron transport chain of acetate-grown Methanosarcina acetivorans ................................................................................ 103 3.1 Abstract ........................................................................................................... 103 3.2 Introduction ..................................................................................................... 104 3.3 Methods and materials .................................................................................... 107 3.4 Results............................................................................................................. 111 3.4.1 Purification of the CdhAE component of the CO dehydrogenase/acetyl-CoA complex (Cdh) of M. acetivorans .............. 111 3.4.2 Properties of the ferredoxin purified from acetate-grown M. acetivorans ............................................................................................. 111 3.4.3 Role of ferredoxin in the membrane-bound electron transport chain ... 116 vi 3.4.4 Role of Rnf in the membrane-bound electron transport chain ............. 116 3.4.5 Role of cytochrome c in the membrane-bound electron transport chain ....................................................................................................... 118 3.4.6 Role of methanophenazine in the membrane-bound electron transport chain. ....................................................................................... 119 3.4.7 Comparative analysis of the M. thermophila genome .......................... 123 3.5 Discussion ....................................................................................................... 125 3.6 Acknowledgements ......................................................................................... 129 3.7 References ....................................................................................................... 130 Chapter 4 Characterization of a novel electron transport protein from Methanosarcina acetivorans ................................................................................ 139 4.1 Abstract ........................................................................................................... 139 4.2 Introduction ..................................................................................................... 140 4.3 Materials and methods .................................................................................... 143 4.4 Results............................................................................................................. 149 4.4.1 Purification and Properties ................................................................... 149 4.4.2 Physiology ............................................................................................ 152 4.4.3 Phylogeny ............................................................................................. 160 4.5 Discussion ....................................................................................................... 160 4.6 Acknowledgements ......................................................................................... 165 4.7 References ....................................................................................................... 165 vii Chapter 5 Discussion and future directions on the understanding of electron transport in acetate-grown Methanosarcina acetivorans ..................................... 171 5.1 A model of energy conservation under fluctuating environmental substrate concentrations ................................................................................. 172 5.2