The Elusive Third Subunit IIa of the Bacterial B-Type Oxidases: The Enzyme from the Hyperthermophile Aquifex aeolicus

The reduction of molecular oxygen to water is catalyzed by complicated membrane-bound metallo-enzymes containing variable numbers of subunits, called cytochrome c oxidases or quinol oxidases. We previously described the cytochrome c oxidase II from the hyperthermophilic bacterium Aquifex aeolicus as a ba3-type two-subunit (subunits I and II) enzyme and showed that it is included in a supercomplex involved in the sulfide-oxygen respiration pathway. It belongs to the B-family of the heme-copper oxidases, enzymes that are far less studied than the ones from family A. Here, we describe the presence in this enzyme of an additional transmembrane helix “subunit IIa”, which is composed of 41 amino acid residues with a measured molecular mass of 5105 Da. Moreover, we show that subunit II, as expected, is in fact longer than the originally annotated protein (from the genome) and contains a transmembrane domain. Using Aquifex aeolicus genomic sequence analyses, N-terminal sequencing, peptide mass fingerprinting and mass spectrometry analysis on entire subunits, we conclude that the B-type enzyme from this bacterium is a three-subunit complex. It is composed of subunit I (encoded by coxA2) of 59000 Da, subunit II (encoded by coxB2) of 16700 Da and subunit IIa which contain 12, 1 and 1 transmembrane helices respectively. A structural model indicates that the structural organization of the complex strongly resembles that of the ba3 cytochrome c oxidase from the bacterium Thermus thermophilus, the IIa helical subunit being structurally the lacking N-terminal transmembrane helix of subunit II present in the A-type oxidases. Analysis of the genomic context of genes encoding oxidases indicates that this third subunit is present in many of the bacterial oxidases from B-family, enzymes that have been described as two-subunit complexes

Les complexes de type bc (aspects fonctionnels et évolution moléculaire)

AIX-MARSEILLE1-BU Sci.St Charles (130552104) / SudocSudocFranceF

Enterococcus faecalis heme-dependent catalase

Enterococcus faecalis cells cannot synthesize porphyrins and do not rely on heme for growth but can take up heme and use it to synthesize heme proteins. We recently described a cytochrome bd in E. faecalis strain V583 and here report the identification of a chromosomal gene, katA, encoding a heme-containing cytoplasmic catalase. The 54-kDa KatA polypeptide shows sequence similarity to members of the family of monofunctional catalases. A hexahistidyl-tagged version of the catalase was purified, and major characteristics of the enzyme were determined. It contains one protoheme IX group per KatA polypeptide. Catalase activity was detected only in E. faecalis cells grown in the presence of heme in the medium; about 2 and 10 muM hemin was required for half-maximal and maximal production of catalase, respectively. Our finding of a catalase whose synthesis is dependent on the acquisition of heme in the opportunistic pathogen E. faecalis might be of clinical importance. Studies of cellular heme transport and heme protein assembly and in vivo synthesis of metalloprotein analogs for biotechnological applications are impeded by the lack of experimental systems. We conclude that the E. faecalis cell potentially provides such a desired system

The three-dimensional structure of catalase from Enterococcus faecalis

Enterococcus faecalis haem catalase was crystallized using lithium sulfate at neutral pH. The crystals belong to space group R3, with unit-cell parameters a = b = 236.9, c = 198.1 Angstrom. The three-dimensional structure was determined by molecular replacement using a subunit of the Proteus mirabilis catalase structure. It was refined against 2.3 Angstrom synchrotron data to a free R factor of 21.8%. Like other catalases, the E. faecalis catalase is a homotetramer with a fold and structure similar to those of its structurally closest relative P. mirabilis. The solvent structure in the active site is identical in the four subunits but differs from that found in other catalases. The structural consequences of the Ramachandran outlier Ser196 are discussed

In vivo production of catalase containing haem analogues.

Haem (protohaem IX) analogues are toxic compounds and have been considered for use as antibacterial agents, but the primary mechanism behind their toxicity has not been demonstrated. Using the haem protein catalase in the Gram-positive bacterium Enterococcus faecalis as an experimental system, we show that a variety of haem analogues can be taken up by bacterial cells and incorporated into haem-dependent enzymes. The resulting cofactor-substituted proteins are dysfunctional, generally resulting in arrested cell growth or death. This largely explains the cell toxicity of haem analogues. In contrast to many other organisms, E. faecalis does not depend on haem for growth, and therefore resists the toxicity of many haem analogues. We have exploited this feature to establish a bacterial in vivo system for the production of cofactor-substituted haem protein variants. As a pilot study, we produced, isolated and analysed novel catalase variants in which the iron atom of the haem prosthetic group is replaced by other metals, i.e. cobalt, gallium, tin, and zinc, and also variants containing meso-protoheme IX, ruthenium meso-protoporphyrin IX and (metal-free) protoporphyrin IX. Engineered haem proteins of this type are of potential use within basic research and the biotechnical industry. Structured digital abstract * MINT-7722358, MINT-7722368: katA (uniprotkb:Q834P5) and katA (uniprotkb:Q834P5) physically interact (MI:0915) by copurification (MI:0025)

Clostridial whole cell and enzyme systems for hydrogen production: current state and perspectives

International audienceStrictly anaerobic bacteria of the Clostridium genus have attracted great interest as potential cell factories for molecular hydrogen production purposes. In addition to being a useful approach to this process, dark fermentation has the advantage of using the degradation of cheap agricultural residues and industrial wastes for molecular hydrogen production. However, many improvements are still required before large-scale hydrogen production from clostridial metabolism is possible. Here we review the literature on the basic biological processes involved in clostridial hydrogen production, and present the main advances obtained so far in order to enhance the hydrogen productivity, as well as suggesting some possible future prospects

Hydrogenases and H2 metabolism in sulfate-reducing bacteria of the Desulfovibrio genus

International audienceHydrogen metabolism plays a central role in sulfate-reducing bacteria of the Desulfovibrio genus and is based on hydrogenases that catalyze the reversible conversion of protons into dihydrogen. These metabolically versatile microorganisms possess a complex hydrogenase system composed of several enzymes of both [FeFe]- and [NiFe]-type that can vary considerably from one Desulfovibrio species to another. This review covers the molecular and physiological aspects of hydrogenases and H2 metabolism in Desulfovibrio but focuses particularly on our model bacterium Desulfovibrio fructosovorans. The search of hydrogenase genes in more than 30 sequenced genomes provides an overview of the distribution of these enzymes in Desulfovibrio. Our discussion will consider the significance of the involvement of electron-bifurcation in H2 metabolism

The dyad of the Y-junction- and a flavin module unites diverse redox enzymes

International audienceThe concomitant presence of two distinctive polypeptide modules, which we have chosen to denominate as the “Y-junction” and the “flavin” module, is observed in 3D structures of enzymes as functionally diverse as complex I, NAD(P)-dependent [NiFe]-hydrogenases and NAD(P)-dependent formate dehydrogenases. Amino acid sequence conservation furthermore suggests that both modules are also part of NAD(P)-dependent [FeFe]-hydrogenases for which no 3D structure model is available yet. The flavin module harbours the site of interaction with the substrate NAD(P) which exchanges two electrons with a strictly conserved flavin moiety. The Y-junction module typically contains four iron-sulphur centres arranged to form a Y-shaped electron transfer conduit and mediates electron transfer between the flavin module and the catalytic units of the respective enzymes. The Y-junction module represents an electron transfer hub with three potential electron entry/exit sites. The pattern of specific redox centres present both in the Y-junction and the flavin module is correlated to present knowledge of these enzymes' functional properties. We have searched publicly accessible genomes for gene clusters containing both the Y-junction and the flavin module to assemble a comprehensive picture of the diversity of enzymes harbouring this dyad of modules and to reconstruct their phylogenetic relationships. These analyses indicate the presence of the dyad already in the last universal common ancestor and the emergence of complex I's EFG-module out of a subgroup of NAD(P)- dependent formate dehydrogenases