Dataset Information

Structural Bases for the Regulation of CO Binding in the Archaeal Protoglobin from Methanosarcina acetivorans.

ABSTRACT: Studies of CO ligand binding revealed that two protein states with different ligand affinities exist in the protoglobin from Methanosarcina acetivorans (in MaPgb*, residue Cys(E20)101 was mutated to Ser). The switch between the two states occurs upon the ligation of MaPgb*. In this work, site-directed mutagenesis was used to explore the role of selected amino acids in ligand sensing and stabilization and in affecting the equilibrium between the "more reactive" and "less reactive" conformational states of MaPgb*. A combination of experimental data obtained from electronic and resonance Raman absorption spectra, CO ligand-binding kinetics, and X-ray crystallography was employed. Three amino acids were assigned a critical role: Trp(60)B9, Tyr(61)B10, and Phe(93)E11. Trp(60)B9 and Tyr(61)B10 are involved in ligand stabilization in the distal heme pocket; the strength of their interaction was reflected by the spectra of the CO-ligated MaPgb* and by the CO dissociation rate constants. In contrast, Phe(93)E11 is a key player in sensing the heme-bound ligand and promotes the rotation of the Trp(60)B9 side chain, thus favoring ligand stabilization. Although the structural bases of the fast CO binding rate constant of MaPgb* are still unclear, Trp(60)B9, Tyr(61)B10, and Phe(93)E11 play a role in regulating heme/ligand affinity.

SUBMITTER: Tilleman L

PROVIDER: S-EPMC4457829 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Structural Bases for the Regulation of CO Binding in the Archaeal Protoglobin from Methanosarcina acetivorans.

Tilleman Lesley L Abbruzzetti Stefania S Ciaccio Chiara C De Sanctis Giampiero G Nardini Marco M Pesce Alessandra A Desmet Filip F Moens Luc L Van Doorslaer Sabine S Bruno Stefano S Bolognesi Martino M Ascenzi Paolo P Coletta Massimo M Viappiani Cristiano C Dewilde Sylvia S

PloS one 20150605 6

Studies of CO ligand binding revealed that two protein states with different ligand affinities exist in the protoglobin from Methanosarcina acetivorans (in MaPgb*, residue Cys(E20)101 was mutated to Ser). The switch between the two states occurs upon the ligation of MaPgb*. In this work, site-directed mutagenesis was used to explore the role of selected amino acids in ligand sensing and stabilization and in affecting the equilibrium between the "more reactive" and "less reactive" conformational ...[more]

PMID: 26047471

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Project description:UnlabelledResults are presented supporting a regulatory role for the product of the MA3302 gene locus (designated MreA) previously annotated as a hypothetical protein in the methanogenic species Methanosarcina acetivorans of the domain Archaea. Sequence analysis of MreA revealed identity to the TrmB family of transcription factors, albeit the sequence is lacking the sensor domain analogous to TrmBL2, abundant in nonmethanogenic species of the domain Archaea. Transcription of mreA was highly upregulated during growth on acetate versus methylotrophic substrates, and an mreA deletion (?mreA) strain was impaired for growth with acetate in contrast to normal growth with methylotrophic substrates. Transcriptional profiling of acetate-grown cells identified 280 genes with altered expression in the ?mreA strain versus the wild-type strain. Expression of genes unique to the acetate pathway decreased whereas expression of genes unique to methylotrophic metabolism increased in the ?mreA strain relative to the wild type, results indicative of a dual role for MreA in either the direct or indirect activation of acetate-specific genes and repression of methylotrophic-specific genes. Gel shift experiments revealed specific binding of MreA to promoter regions of regulated genes. Homologs of MreA were identified in M. acetivorans and other Methanosarcina species for which expression patterns indicate roles in regulating methylotrophic pathways.ImportanceSpecies in the domain Archaea utilize basal transcription machinery resembling that of the domain Eukarya, raising questions addressing the role of numerous putative transcription factors identified in sequenced archaeal genomes. Species in the genus Methanosarcina are ideally suited for investigating principles of archaeal transcription through analysis of the capacity to utilize a diversity of substrates for growth and methanogenesis. Methanosarcina species switch pathways in response to the most energetically favorable substrate, metabolizing methylotrophic substrates in preference to acetate marked by substantial regulation of gene expression. Although conversion of the methyl group of acetate accounts for most of the methane produced in Earth's biosphere, no proteins involved in the regulation of genes in the acetate pathway have been reported. The results presented here establish that MreA participates in the global regulation of diverse methanogenic pathways in the genus Methanosarcina. Finally, the results contribute to a broader understanding of transcriptional regulation in the domain Archaea.

Project description:BackgroundAcetate is the major source of methane in nature. The majority of investigations have focused on acetotrophic methanogens for which energy-conserving electron transport is dependent on the production and consumption of H₂ as an intermediate, although the great majority of acetotrophs are unable to metabolize H₂. The presence of cytochrome c and a complex (Ma-Rnf) homologous to the Rnf (Rhodobacter nitrogen fixation) complexes distributed in the domain Bacteria distinguishes non-H₂-utilizing Methanosarcina acetivorans from H₂-utilizing species suggesting fundamentally different electron transport pathways. Thus, the membrane-bound electron transport chain of acetate-grown M. acetivorans was investigated to advance a more complete understanding of acetotrophic methanogens.ResultsA component of the CO dehydrogenase/acetyl-CoA synthase (CdhAE) was partially purified and shown to reduce a ferredoxin purified using an assay coupling reduction of the ferredoxin to oxidation of CdhAE. Mass spectrometry analysis of the ferredoxin identified the encoding gene among annotations for nine ferredoxins encoded in the genome. Reduction of purified membranes from acetate-grown cells with ferredoxin lead to reduction of membrane-associated multi-heme cytochrome c that was re-oxidized by the addition of either the heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB) or 2-hydoxyphenazine, the soluble analog of methanophenazine (MP). Reduced 2-hydoxyphenazine was re-oxidized by membranes that was dependent on addition of CoM-S-S-CoB. A genomic analysis of Methanosarcina thermophila, a non-H2-utilizing acetotrophic methanogen, identified genes homologous to cytochrome c and the Ma-Rnf complex of M. acetivorans.ConclusionsThe results support roles for ferredoxin, cytochrome c and MP in the energy-conserving electron transport pathway of non-H₂-utilizing acetotrophic methanogens. This is the first report of involvement of a cytochrome c in acetotrophic methanogenesis. The results suggest that diverse acetotrophic Methanosarcina species have evolved diverse membrane-bound electron transport pathways leading from ferredoxin and culminating with MP donating electrons to the heterodisulfide reductase (HdrDE) for reduction of CoM-S-S-CoB.

Dataset Information

Structural Bases for the Regulation of CO Binding in the Archaeal Protoglobin from Methanosarcina acetivorans.

Publications

Structural Bases for the Regulation of CO Binding in the Archaeal Protoglobin from Methanosarcina acetivorans.

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