Energy-converting [NiFe] hydrogenases in archaea and bacteria: insights into the energy-transducing mechanism

In recent years a group of multisubunit membrane-bound [NiFe] hydrogenases has been identified in a variety of anaerobic or facultative anaerobic microorganisms. These enzymes share two conserved integral membrane proteins and four conserved hydrophilic proteins with the energy-conserving NADH:quinone oxidoreductases (complex I). Based on experimental evidence derived from physiological and biochemical studies, the various members of this hydrogenase family have been proposed to function as ion pumps being involved in energy-conserving electron transport, reverse electron transport, or both. Therefore these enzymes have been designated energy-converting [NiFe] hydrogenases. In the present work the energy transducing mechanism of energy-converting hydrogenases was studied in comparison to complex I. A particular attention was given to the prosthetic groups involved in the electron transfer pathway, the role of the membrane part and the identification of the coupling ion used by these enzymes. The majority of the experiments were carried out with Ech hydrogenase from Methanosarcina barkeri. The sequence of Ech hydrogenase predicts the binding of three [4Fe-4S] clusters, one by subunit EchC and two by subunit EchF. Previous studies had shown that two of these clusters could be fully reduced under 1 bar of H2 at pH 7 giving rise to two distinct S ½ EPR signals, designated as the g = 1.89 and the g = 1.92 signal. Redox titrations at different pH values demonstrated that these two clusters had a pH-dependent midpoint potential indicating a function in ion pumping. To assign these EPR signals to the subunits of the enzyme a set of M. barkeri mutants was generated in which seven of eight conserved cysteine residues in EchF were individually replaced by serine. EPR spectra recorded from the isolated mutant enzymes revealed a strong reduction or complete loss of the g = 1.92 signal whereas the g =1.89 signal was still detectable as the major EPR signal in five mutant enzymes. It is concluded that the cluster giving rise to the g = 1.89 signal is the proximal cluster located in EchC and that the g = 1.92 signal results from one of the clusters of subunit EchF. The pH-dependent midpoint potential of these two [4Fe-4S] clusters suggests that these clusters simultaneously mediate electron and ion transfer and thus could be an essential part of the ion-translocating machinery. In the two integral membrane subunits of Ech carboxylic residues are found that are highly conserved within the family of energy-converting hydrogenases and complex I. These residues could be part of a transmembrane ion channel. In line with this, Ech hydrogenase activity was inhibited by the carboxyl-modifying reagent N,N’–dicyclohexylcarbodiimide (DCCD). The inhibition of the enzyme correlated quite well with the incorporation of [14C]DCCD in subunits EchA. Using a combination of FT-IR difference spectroscopy and electrochemistry it was shown that the electron transfer reaction catalyzed by Ech hydrogenase from M. barkeri induces a conformational change of the enzyme and the protonation of amino acid side chains. Oxidized minus reduced spectra in the mid infrared range (1800 to 1200 cm-1) revealed conformational changes in the amide I region and a signal at 1720 cm-1 attributed to either an Asp or Glu side chain, protonated in the oxidized state. To identify the coupling ion used by energy converting hydrogenases studies with the enzyme from Carboxydothermus hydrogenoformans were performed. Cell suspensions of C. hydrogenoformans were found to couple the oxidation of CO to CO2 and H2 with the translocation of protons across the membrane at pH 5.9. This transient acidification was inhibited by the protonophore CCCP but was not affected by the sodium ionophore ETH-157, indicating the generation of a primary electrochemical proton gradient. However, no proton translocation coupled to CO oxidation was observed at pH 6.7. On the other hand, at neutral pH, CO oxidation was coupled to sodium ion translocation. This reaction was protonophore insensitive, indicating a primary Na+ translocation. These data indicate that the Coo hydrogenase from C. hydrogenoformans could be a primary sodium pump, which may also use H+ at low pH

Energy-converting [NiFe] hydrogenases in archaea and bacteria: insights into the energy-transducing mechanism

In recent years a group of multisubunit membrane-bound [NiFe] hydrogenases has been identified in a variety of anaerobic or facultative anaerobic microorganisms. These enzymes share two conserved integral membrane proteins and four conserved hydrophilic proteins with the energy-conserving NADH:quinone oxidoreductases (complex I). Based on experimental evidence derived from physiological and biochemical studies, the various members of this hydrogenase family have been proposed to function as ion pumps being involved in energy-conserving electron transport, reverse electron transport, or both. Therefore these enzymes have been designated energy-converting [NiFe] hydrogenases. In the present work the energy transducing mechanism of energy-converting hydrogenases was studied in comparison to complex I. A particular attention was given to the prosthetic groups involved in the electron transfer pathway, the role of the membrane part and the identification of the coupling ion used by these enzymes. The majority of the experiments were carried out with Ech hydrogenase from Methanosarcina barkeri. The sequence of Ech hydrogenase predicts the binding of three [4Fe-4S] clusters, one by subunit EchC and two by subunit EchF. Previous studies had shown that two of these clusters could be fully reduced under 1 bar of H2 at pH 7 giving rise to two distinct S ½ EPR signals, designated as the g = 1.89 and the g = 1.92 signal. Redox titrations at different pH values demonstrated that these two clusters had a pH-dependent midpoint potential indicating a function in ion pumping. To assign these EPR signals to the subunits of the enzyme a set of M. barkeri mutants was generated in which seven of eight conserved cysteine residues in EchF were individually replaced by serine. EPR spectra recorded from the isolated mutant enzymes revealed a strong reduction or complete loss of the g = 1.92 signal whereas the g =1.89 signal was still detectable as the major EPR signal in five mutant enzymes. It is concluded that the cluster giving rise to the g = 1.89 signal is the proximal cluster located in EchC and that the g = 1.92 signal results from one of the clusters of subunit EchF. The pH-dependent midpoint potential of these two [4Fe-4S] clusters suggests that these clusters simultaneously mediate electron and ion transfer and thus could be an essential part of the ion-translocating machinery. In the two integral membrane subunits of Ech carboxylic residues are found that are highly conserved within the family of energy-converting hydrogenases and complex I. These residues could be part of a transmembrane ion channel. In line with this, Ech hydrogenase activity was inhibited by the carboxyl-modifying reagent N,N’–dicyclohexylcarbodiimide (DCCD). The inhibition of the enzyme correlated quite well with the incorporation of [14C]DCCD in subunits EchA. Using a combination of FT-IR difference spectroscopy and electrochemistry it was shown that the electron transfer reaction catalyzed by Ech hydrogenase from M. barkeri induces a conformational change of the enzyme and the protonation of amino acid side chains. Oxidized minus reduced spectra in the mid infrared range (1800 to 1200 cm-1) revealed conformational changes in the amide I region and a signal at 1720 cm-1 attributed to either an Asp or Glu side chain, protonated in the oxidized state. To identify the coupling ion used by energy converting hydrogenases studies with the enzyme from Carboxydothermus hydrogenoformans were performed. Cell suspensions of C. hydrogenoformans were found to couple the oxidation of CO to CO2 and H2 with the translocation of protons across the membrane at pH 5.9. This transient acidification was inhibited by the protonophore CCCP but was not affected by the sodium ionophore ETH-157, indicating the generation of a primary electrochemical proton gradient. However, no proton translocation coupled to CO oxidation was observed at pH 6.7. On the other hand, at neutral pH, CO oxidation was coupled to sodium ion translocation. This reaction was protonophore insensitive, indicating a primary Na+ translocation. These data indicate that the Coo hydrogenase from C. hydrogenoformans could be a primary sodium pump, which may also use H+ at low pH

E-Business and Entrepreneurial Cooperation--A New Customer-Oriented E-Business Modeling Approach Validated in the Case of a Collaboration Network in the German Manufacturing Industry

Nowadays, the Internet and web-based E-Business solutions play a crucial enabling role for the design and implementation of new Business Models. This implies high chances, but also remarkable risks for enterprises that have to face choice and adoption of a Business Model. As a matter of fact, the development and implementation of a strategically not appropriate Business Model would crucially undermine the long-term success of a company in the global market arena. Therefore the clear need for action in the field of methodical Business Modeling. Our contribution presents a new approach for a customeroriented (E-)Business Modeling [7], with a specific attention on entrepreneurial cooperation. The approach had been validated for a collaboration network in the German manufacturing industry

Радиационный контроль в современных процессах нефтедобычи

Рассмотрены вопросы радиоэкологического контроля на нефтедобывающих предприятиях. Показана возможность использования программных продуктов для обеспечения комплексной оценки радиационной обстановки на объектах и территориях нефтедобывающих предприятий. Освещены основные аспекты взаимодействия научно-исследовательских лабораторий с Центрами экологической безопасности предприятий и их значение для эффективного ресурсосбережения в современных процессах нефтедобычи

Electronic Business in Germany: Current Challenges and Future Perspectives - Results of an Explorative Survey -

The objective of the present contribution is to present the results of an explorative survey in the field of E-Business conducted with German enterprises at the end of the year 2003. The survey aimed to convey a picture of the present stage as well the future trends of E-Business in the German entrepreneurial world. An analysis of the present and future developments of E-Business as well as of the related organisational, technical and human implications for enterprises will be presented. Limitations and open challenges of research as well as the planned further work will be eventually discussed

A Methodology to Support the Design and Deployment of Knowledge Management within Inter-organizational Networks

A well-functioning Knowledge Management is a competitive advantage for enterprises that act in co-operative and distributed networks with knowledge intensive production processes. A Knowledge Management approach that integrates both, hard factors (e.g., Information Technology) and soft factors (e.g., cultural aspects) for distributed and dynamic entrepreneurial (inter-organisational) networks is currently missing. This paper presents research findings of a project that is developing a methodology to support a service provider responsible for the KM within distributed entrepreneurial networks

A Holistic Approach for E-Business Engineering

In the recent years, the broader application of web-based technologies caused radical changes and a consequent rapid development within the entrepreneurial environment. In order to exploit first-mover advantages, enterprises often preferred a quick-paced introduction of E-Business solutions, hence neglecting more holistic and integrated approaches. This fact implied that E-Business solutions were usually simply and hastily embedded into the existing business processes and organizational structures. As a result, E-Business projects often did not reach the striven targets or even failed, with the consequently growing lack of trust towards the above-mentioned business approach. Hence, there is a clear need for action in the field of methodical development, deployment and integration of E-Business solutions into the entrepreneurial structure. We present an integrated framework for the engineering of E-Business, which is the result of a 3 year experience at FIR

Overproduction of the membrane-bound [NiFe]-hydrogenase in Thermococcus kodakarensis and its effect on hydrogen production

The hyperthermophilic archaeon Thermococcus kodakarensis can utilize sugars or pyruvate for growth. In the absence of elemental sulfur, the electrons via oxidation of these substrates are accepted by protons, generating molecular hydrogen (H2). The hydrogenase responsible for this reaction is a membrane-bound [NiFe]-hydrogenase (Mbh). In this study, we have examined several possibilities to increase the protein levels of Mbh in T. kodakarensis by genetic engineering. Highest levels of intracellular Mbh levels were achieved when the promoter of the entire mbh operon (TK2080-TK2093) was exchanged to a strong constitutive promoter from the glutamate dehydrogenase gene (TK1431) (strain MHG1). When MHG1 was cultivated under continuous culture conditions using pyruvate-based medium, a nearly 25% higher specific hydrogen production rate (SHPR) of 35.3 mmol H2 g-dcw-1 h-1 was observed at a dilution rate of 0.31 h-1. We also combined mbh overexpression using an even stronger constitutive promoter from the cell surface glycoprotein gene (TK0895) with disruption of the genes encoding the cytosolic hydrogenase (Hyh) and an alanine aminotransferase (AlaAT), both of which are involved in hydrogen consumption (strain MAH1). At a dilution rate of 0.30 h-1, the SHPR was 36.2 mmol H2 g-dcw-1 h-1, corresponding to a 28% increase compared to that of the host T. kodakarensis strain. Increasing the dilution rate to 0.83 h-1 or 1.07 h-1 resulted in a SHPR of 120 mmol H2 g-dcw-1 h-1, which is one of the highest production rates observed in microbial fermentation

Biosynthesis of Salmonella enterica [NiFe]-hydrogenase-5 : probing the roles of system-specific accessory proteins

A subset of bacterial [NiFe]-hydrogenases have been shown to be capable of activating dihydrogen-catalysis under aerobic conditions; however, it remains relatively unclear how the assembly and activation of these enzymes is carried out in the presence of air. Acquiring this knowledge is important if a generic method for achieving production of O2-resistant [NiFe]-hydrogenases within heterologous hosts is to be developed. Salmonella enterica serovar Typhimurium synthesizes the [NiFe]-hydrogenase-5 (Hyd-5) enzyme under aerobic conditions. As well as structural genes, the Hyd-5 operon also contains several accessory genes that are predicted to be involved in different stages of biosynthesis of the enzyme. In this work, deletions in the hydF, hydG, and hydH genes have been constructed. The hydF gene encodes a protein related to Ralstonia eutropha HoxO, which is known to interact with the small subunit of a [NiFe]-hydrogenase. HydG is predicted to be a fusion of the R. eutropha HoxQ and HoxR proteins, both of which have been implicated in the biosynthesis of an O2-tolerant hydrogenase, and HydH is a homologue of R. eutropha HoxV, which is a scaffold for [NiFe] cofactor assembly. It is shown here that HydG and HydH play essential roles in Hyd-5 biosynthesis. Hyd-5 can be isolated and characterized from a ΔhydF strain, indicating that HydF may not play the same vital role as the orthologous HoxO. This study, therefore, emphasises differences that can be observed when comparing the function of hydrogenase maturases in different biological systems

Herausforderungen und Erfolgsfaktoren für das Wissensmanagement in verteilten, wissensintensiven Unternehmensnetzwerken – Ausgewählte Ergebnisse einer explorativen Umfrage

In den letzten Jahren verstärkte sich der Trend hin zu mehr Kooperationen in vernetzten Strukturen. Hintergrund dieser Entwicklung ist einerseits die Konzentration vieler Unternehmen auf ihre Kernkompetenzen, welche die Auslagerung vieler Funktionen zur Folge hat. Andererseits werden auch große Geschäftsbereiche in kleinere Einheiten aufgeteilt, um flexibler auf sich immer schneller verändernde Kundenanforderungen reagieren zu können. Dieser höheren Flexibilität steht jedoch eine steigende Notwendigkeit zur erfolgreichen Kooperation über Bereichs- und Unternehmensgrenzen hinweg gegenüber. War Wissen bisher schon als Erfolgsfaktor in Unternehmen von großer Bedeutung für die Wettbewerbsfähigkeit auf globalen Märkten, gilt dies für Netzwerke um so mehr, da der zielgerichtete Wissenstransfer zwischen den beteiligten Partnern eine wesentliche Voraussetzung für eine erfolgreiche Kooperation darstellt. Allerdings erschweren eine Reihe netzwerkspezifischer Probleme ein effizientes und effektives Wissensmanagement (WM). Insbesondere in wissensintensiven Kooperationen führen unterschiedliche Ziel- und Wertsysteme dazu, dass der Austausch von Wissen oft an kulturellen Barrieren und mangelndem Vertrauen zwischen den Partnern scheitert. Eine Reihe von Arbeiten hat sich bisher mit dem Thema Wissensmanagement beschäftigt. Sie beschränken sich jedoch meist auf Ansätze in fest definierten Unternehmensgrenzen und sind zudem stark technologiefokussiert. Erste Ansätze im Bereich des Wissensmanagements in Netzwerken beleuchten insgesamt gesehen viele relevante Aspekte des hier behandelten Problems. Allerdings werden diese Aspekte in keiner der Arbeiten durch eine ganzheitliche Betrachtung integriert. Adäquate Modelle und Methoden, die einer solchen ganzheitlichen Betrachtung gerecht werden, fehlen bislang