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One-megadalton metalloenzyme complex in Geobacter metallireducens involved in benzene ring reduction beyond the biological redox window.

ABSTRACT: Reversible biological electron transfer usually occurs between redox couples at standard redox potentials ranging from +0.8 to -0.5 V. Dearomatizing benzoyl-CoA reductases (BCRs), key enzymes of the globally relevant microbial degradation of aromatic compounds at anoxic sites, catalyze a biological Birch reduction beyond the negative limit of this redox window. The structurally characterized BamBC subunits of class II BCRs accomplish benzene ring reduction at an active-site tungsten cofactor; however, the mechanism and components involved in the energetic coupling of endergonic benzene ring reduction have remained hypothetical. We present a 1-MDa, membrane-associated, Bam[(BC)₂DEFGHI]₂ complex from the anaerobic bacterium Geobacter metallireducens harboring 4 tungsten, 4 zinc, 2 selenocysteines, 6 FAD, and >50 FeS cofactors. The results suggest that class II BCRs catalyze electron transfer to the aromatic ring, yielding a cyclic 1,5-dienoyl-CoA via two flavin-based electron bifurcation events. This work expands our knowledge of energetic couplings in biology by high-molecular-mass electron bifurcating machineries.

SUBMITTER: Huwiler SG

PROVIDER: S-EPMC6369795 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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One-megadalton metalloenzyme complex in <i>Geobacter metallireducens</i> involved in benzene ring reduction beyond the biological redox window.

Huwiler Simona G SG Löffler Claudia C Anselmann Sebastian E L SEL Stärk Hans-Joachim HJ von Bergen Martin M Flechsler Jennifer J Rachel Reinhard R Boll Matthias M

Proceedings of the National Academy of Sciences of the United States of America 20190123 6

Reversible biological electron transfer usually occurs between redox couples at standard redox potentials ranging from +0.8 to -0.5 V. Dearomatizing benzoyl-CoA reductases (BCRs), key enzymes of the globally relevant microbial degradation of aromatic compounds at anoxic sites, catalyze a biological Birch reduction beyond the negative limit of this redox window. The structurally characterized BamBC subunits of class II BCRs accomplish benzene ring reduction at an active-site tungsten cofactor; ho ...[more]

PMID: 30674680

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Project description:Anaerobic activation of benzene is expected to represent a novel biochemistry of environmental significance but research into the mechanisms has been stymied by a lack of a genetically tractable pure culture which unequivocally does not use molecular oxygen to activate benzene. Geobacter metallireducens grew in a medium in which benzene was the sole electron donor and Fe(III) was the sole electron acceptor with a stoichiometry of benzene loss and Fe(III) reduction consistent with benzene oxidation to carbon dioxide coupled with Fe(III) reduction. Phenol labeled with 18O was produced when the medium was labeled with H218O, as expected for a true anaerobic conversion of benzene to phenol. Gene expression patterns indicated that benzene was metabolized through a phenol intermediate rather than benzoate or toluene. Deletion of ppcB, which encodes a subunit of the phenylphosphate carboxylase, an enzyme required for phenol metabolism, inhibited metabolism of benzene. Deleting genes specific for benzoate or toluene metabolism did not. Comparison of gene expression patterns in cells grown on benzene versus cells grown on phenol revealed genes specifically expressed in benzene-grown cells. Deletion of one of these, Gmet_3376, inhibited anaerobic benzene oxidation, but not the metabolism of phenol, benzoate, or toluene. The availability of a genetically tractable pure culture that can anaerobically convert benzene to phenol with oxygen derived from water should significantly accelerate elucidation of the mechanisms by which benzene can be activated in the absence of molecular oxygen. Total RNA from three separate cultures of G. metallireducens grown with 250 ÂµM benzene three separate cultures of G. metallireducens grown with 500 ÂµM phenol three separate cultures of G. metallireducens grown with 1 mM benzoate three separate cultures of G. metallireducens grown with 500 ÂµM toluene three separate cultures of G. metallireducens grown with 10 mM acetate were used to study [1] Anaerobic oxidation of benzene by G. metallireducens (Benzene vs. acetate, Benzene vs. benzoate, Benzene vs. phenol, Benzene vs. toluene) [2] Anaerobic oxidation of benzoate by G. metallireducens (Benzoate vs. acetate) [3] Anaerobic oxidation of phenol by G. metallireducens (Phenol vs. acetate) [4] Anaerobic oxidation of toluene by G. metallireducens (Toluene vs. acetate) Each chip measures the expression level of 3,627 genes from G. metallireducens DSM 7210 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

Project description:Electrogenic bacteria, such as Geobacter, can couple the oxidation of carbon sources to the reduction of extracellular electron acceptors; such acceptors include toxic and radioactive metals, as well as electrode surfaces, making Geobacter a suitable candidate for applied use in bioremediation and bioenergy generation. Geobacter metallireducens is more promising in this regard than the better studied Geobacter sulfurreducens, as it has more efficient Fe (III) reduction rates and can convert nitrate to ammonia. The operon responsible for nitrate reductase activity in G. metallireducens includes the gene encoding the cytochrome PpcF, which was proposed to exchange electrons with nitrate reductase. In the present work, we perform a biochemical and a biophysical characterization of PpcF. Spectroscopic techniques, including circular dichroism (CD), UV-visible, and nuclear magnetic resonance (NMR), revealed that the cytochrome is very stable (T m > 85 °C), contains three low-spin hemes, and is diamagnetic (S = 0) and paramagnetic (S = 1/2) in the reduced and oxidized states, respectively. The NMR chemical shifts of the heme substituents were assigned and used to determine the heme core architecture of PpcF. Compared to the PpcA-family from G. sulfurreducens, the spatial disposition of the hemes is conserved, but the functional properties are clearly distinct. In fact, potentiometric titrations monitored by UV-visible absorption reveal that the reduction potential values of PpcF are significantly less negative (-56 and -64 mV, versus the normal hydrogen electrode at pH 7.0 and 8.0, respectively). NMR redox titrations showed that the order of oxidation of the hemes is IV-I-III, a feature not observed for G. sulfurreducens. The different redox properties displayed by PpcF, including the small redox-Bohr effect and low reduction potential value of heme IV, were structurally rationalized and attributed to the lower number of positively charged residues located in the vicinity of heme IV. Overall, the redox features of PpcF suggest that biotechnological applications of G. metallireducens may require less negative working functional redox windows than those using by G. sulfurreducens.

Project description:The ability of Geobacter species to readily donate electrons to extracellular electron acceptors makes the study of their physiology not only important for the understanding of environmental processes, but also for industrial applications such as bioelectronics and electrosynthesis. Studies in G. sulfurreducens have shown that outer surface components, such as c-type cytochromes and conductive type IV pili play an important role in direct electron transfer to extracellular electron acceptors such as Fe(III) oxides and electrodes. However, many of these thoroughly studied outer surface components, including c-type cytochromes, are not well conserved among Geobacter species. In order to better understand which components are involved in extracellular electron transfer in Geobacter species other than G. sulfurreducens, studies were conducted with its close relative G. metallireducens. Whole-genome microarray analysis revealed that 23 of the 91 putative c-type cytochromes encoded in the G. metallireducens genome were upregulated at least 2-fold in cells grown with Fe(III) oxide compared to cells in which Fe(III) citrate was provided as the terminal electron acceptor. Protein identification with liquid-chromatography/mass spectrometry detected 6 c-type cytochromes that were more abundant in the outer surface cell fraction of cells that were grown with Fe(III) oxide as the terminal electron acceptor compared to cells grown on Fe(III) citrate. 22 genes encoding c-type cytochromes were chosen for gene deletion. Deletion of 6 genes encoding for c-type cytochromes, a gene encoding for a lipopolysaccharide biosynthesis-associated protein, and a gene encoding for a NHL- repeat containing protein inhibited growth when Fe(III) oxide was provided as the electron acceptor. This study suggests that there are different roads for extracellular electron transfer in Geobacteraceae since homologous c-type cytochromes have different functions from one species to the other, and novel components not previously found to be essential for extracellular electron transfer were identified.

Dataset Information

One-megadalton metalloenzyme complex in Geobacter metallireducens involved in benzene ring reduction beyond the biological redox window.

Publications

One-megadalton metalloenzyme complex in <i>Geobacter metallireducens</i> involved in benzene ring reduction beyond the biological redox window.

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