Bruno C. Marreiros

A missing link between complex I and group 4 membrane-bound (NiFe) hydrogenases☆

Complex I of respiratory chains is an energy transducing enzyme present in most bacteria, mitochondria and chloroplasts. It catalyzes the oxidation of NADH and the reduction of quinones, coupled to cation translocation across the membrane. The complex has a modular structure composed of several proteins most of which are identified in other complexes. Close relations between complex I and group 4 membrane-bound [NiFe] hydrogenases and some subunits of multiple resistance to pH (Mrp) Na(+)/H(+) antiporters have been observed before and the suggestion that complex I arose from the association of a soluble nicotinamide adenine dinucleotide (NAD(+)) reducing hydrogenase with a Mrp-like antiporter has been put forward. In this article we performed a thorough taxonomic profile of prokaryotic group 4 membrane-bound [NiFe] hydrogenases, complexes I and complex I-like enzymes. In addition we have investigated the different gene clustering organizations of such complexes. Our data show the presence of complexes related to hydrogenases but which do not contain the binding site of the catalytic centre. These complexes, named before as Ehr (energy-converting hydrogenases related complexes) are a missing link between complex I and group 4 membrane-bound [NiFe] hydrogenases. Based on our observations we put forward a different perspective for the relation between complex I and related complexes. In addition we discuss the evolutionary, functional and mechanistic implications of this new perspective. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.

Decoupling of the catalytic and transport activities of complex I from Rhodothermus marinus by sodium/proton antiporter inhibitor.

The energy transduction by complex I from Rhodothermusmarinus was addressed by studying the influence of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on the activities of this enzyme. EIPA is an inhibitor of both Na(+)/H(+) antiporter and complex I NADH:quinone oxidoreductase activity. We performed studies of NADH:quinone oxidoreductase and H(+) and Na(+) translocation activities of complex I from R. marinus at different concentrations of EIPA, using inside-out membrane vesicles. We observed that the oxidoreductase activity and both H(+) and Na(+) transports are inhibited by EIPA. Most interestingly, the catalytic and the two transport activities showed different inhibition profiles. The transports are inhibited at concentrations of EIPA at which the catalytic activity is not affected. In this way the catalytic and transport activities were decoupled. Moreover, the inhibition of the catalytic activity was not influenced by the presence of Na(+), whereas the transport of H(+) showed different inhibition behaviors in the presence and absence of Na(+). Taken together our observations indicate that complex I from R. marinus performs energy transduction by two different processes: proton pumping and Na(+)/H(+) antiporting. The decoupling of the catalytic and transport activities suggests the involvement of an indirect coupling mechanism, possibly through conformational changes.

Type II NADH:quinone oxidoreductase family: phylogenetic distribution, structural diversity and evolutionary divergences

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins, crucial for the catabolic metabolism, because they contribute to the maintenance of the NADH/NAD+ balance. In several pathogenic bacteria and protists, NDH-2s are the only enzymes performing respiratory NADH:quinone oxidoreductase activity. For this reason and for being considered absent in mammals, NDH-2s were proposed as suitable targets for novel antimicrobial therapies. We selected all sequences of genes encoding NDH-2s from fully sequenced genomes present in the KEGG database. These genes were present in 61% of the 1805 species belonging to Eukarya (83%), Bacteria (60%) and Archaea (32%). Notably sequences from mammal species including humans were retrieved in our selection as NDH-2s. The data obtained and the already available information allowed systematizing several properties of NDH-2s: (i) the existence of additional sequence motifs with putative regulatory functions, (ii) specificity towards NADH or NADPH and (iii) the type of quinone binding motif. We observed that NDH-2 family distribution is not congruent with the taxonomic tree, suggesting different origins for the eukaryotic sequences and possible lateral gene transfer among prokaryotes. We note the absence of genes coding for NDH-2 in anaerobic phyla and the presence of multiple copies in several genomes, specifically in cyanobacteria. These observations inspired us to propose a metabolic hypothesis for the appearance of NDH-2s.

The role of proton and sodium ions in energy transduction by respiratory complex I

Respiratory complex I plays a central role in energy transduction. It catalyzes the oxidation of NADH and the reduction of quinone, coupled to cation translocation across the membrane, thereby establishing an electrochemical potential. For more than half a century, data on complex I has been gathered, including recently determined crystal structures, yet complex I is the least understood complex of the respiratory chain. The mechanisms of quinone reduction, charge translocation and their coupling are still unknown. The H+ is accepted to be the coupling ion of the system; however, Na+ has also been suggested to perform such a role. In this article, we address the relation of those two ions with complex I and refer ion pump and Na+/H+ antiporter as possible transport mechanisms of the system. We put forward a hypothesis to explain some apparently contradictory data on the nature of the coupling ion, and we revisit the role of H+ and Na+ cycles in the overall bioenergetics of the cell.

The antiporter-like subunit constituent of the universal adaptor of complex I, group 4 membrane-bound [NiFe]-hydrogenases and related complexes

Abstract We have recently investigated the long-recognized relationship between complex I and group 4 [NiFe] hydrogenases and we have established the so-called Energy-converting hydrogenase related (Ehr) complex as a new member of the family. We have also observed that four subunits, homologues to NuoB, D, H and L, are common to the members of the family. We have designated this common group of subunits the universal adaptor. Taking into account the similarity of the Na+/H+ antiporter-like subunits of complex I (NuoL, NuoM and NuoN) and the unique structural characteristic of the long amphipathic α helix part of NuoL, the nature of the antiporter-like subunit of the universal adaptor was questioned. Thus, in this work we further explore the properties of the universal adaptor, investigating which antiporter-like subunit is part of the universal adaptor. We observe that the universal adaptor contains an antiporter-like subunit with a long amphipathic α helix, similar to NuoL. Consequently, the long helix is a common denominator that has been conserved in all members of the family. Such conservation surely reflects the key role of such helix in the energy transduction mechanism of this family of enzymes.

Structural and Functional insights into the catalytic mechanism of the Type II NADH:quinone oxidoreductase family

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains. These proteins contribute indirectly to the establishment of the transmembrane difference of electrochemical potential by catalyzing the reduction of quinone by oxidation of NAD(P)H. NDH-2s are widespread enzymes being present in the three domains of life. In this work, we explored the catalytic mechanism of NDH-2 by investigating the common elements of all NDH-2s, based on the rationale that conservation of such elements reflects their structural/functional importance. We observed conserved sequence motifs and structural elements among 1762 NDH-2s. We identified two proton pathways possibly involved in the protonation of the quinone. Our results led us to propose the first catalytic mechanism for NDH-2 family, in which a conserved glutamate residue, E172 (in NDH-2 from Staphylococcus aureus) plays a key role in proton transfer to the quinone pocket. This catalytic mechanism may also be extended to the other members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, such as sulfide:quinone oxidoreductases.

Study of ion translocation by respiratory complex I. A new insight using 23Na NMR spectroscopy

The research on complex I has gained recently a new enthusiasm, especially after the resolution of the crystallographic structures of bacterial and mitochondrial complexes. Most attention is now dedicated to the investigation of the energy coupling mechanism(s). The proton has been identified as the coupling ion, although in the case of some bacterial complexes I Na(+) has been proposed to have that role. We have addressed the relation of some complexes I with Na(+) and developed an innovative methodology using (23)Na NMR spectroscopy. This allowed the investigation of Na(+) transport taking the advantage of directly monitoring changes in Na(+) concentration. Methodological aspects concerning the use of (23)Na NMR spectroscopy to measure accurately sodium transport in bacterial membrane vesicles are discussed here. External-vesicle Na(+) concentrations were determined by two different methods: 1) by integration of the resonance frequency peak and 2) using calibration curves of resonance frequency shift dependence on Na(+) concentration. Although the calibration curves are a suitable way to determine Na(+) concentration changes under conditions of fast exchange, it was shown not to be applicable to the bacterial membrane vesicle systems. In this case, the integration of the resonance frequency peak is the most appropriate analysis for the quantification of external-vesicle Na(+) concentration. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Respiratory complex I from Escherichia coli does not transport Na(+) in the absence of its NuoL subunit.

We investigated H+ and Na+ transport by complex I from Escherichia coli devoid of the NuoL subunit, which is probably part of the ion translocating machinery. We observed that complex I devoid of the NuoL subunit still translocates H+, although to a smaller extension than the complete version of complex I, but does not transport Na+. Our results unequivocally reinforce the observation that E. coli complex I transports Na + in the opposite direction to that of the H+ and show that NuoL subunit is involved in the translocation of both ions by complex I.

Taxonomic distribution, structure/function relationship and metabolic context of the two families of sulfide dehydrogenases: SQR and FCSD

Hydrogen sulfide (H2S) is a versatile molecule with different functions in living organisms: it can work as a metabolite of sulfur and energetic metabolism or as a signaling molecule in higher Eukaryotes. H2S is also highly toxic since it is able to inhibit heme cooper oxygen reductases, preventing oxidative phosphorylation. Due to the fact that it can both inhibit and feed the respiratory chain, the immediate role of H2S on energy metabolism crucially relies on its bioavailability, meaning that studying the central players involved in the H2S homeostasis is key for understanding sulfide metabolism. Two different enzymes with sulfide oxidation activity (sulfide dehydrogenases) are known: flavocytochrome c sulfide dehydrogenase (FCSD), a sulfide:cytochrome c oxidoreductase; and sulfide:quinone oxidoreductase (SQR). In this work we performed a thorough bioinformatic study of SQRs and FCSDs and integrated all published data. We systematized several properties of these proteins: (i) nature of flavin binding, (ii) capping loops and (iii) presence of key amino acid residues. We also propose an update to the SQR classification system and discuss the role of these proteins in sulfur metabolism.