Energy-converting [NiFe] hydrogenases: more than just H2 activation

The well-characterized [NiFe] hydrogenases have a key function in the H2 metabolism of various microorganisms. A subfamily of the [NiFe] hydrogenases with unique properties has recently been identified. The six conserved subunits that build the core of these membrane-bound hydrogenases share sequence similarity with subunits that form the catalytic core of energy-conserving NADH:quinone oxidoreductases (complex I). The physiological role of some of these hydrogenases is to catalyze the reduction of H+ with electrons derived from reduced ferredoxins or polyferredoxins. This exergonic reaction is coupled to energy conservation by means of electron-transport phosphorylation. Other members of this hydrogenase subfamily mainly function in providing the cell with reduced ferredoxin using H2 as electron donor in a reaction driven by reverse electron transport. These hydrogenases have therefore been designated as energy-converting [NiFe] hydrogenases.     

NADH formation by Na+-coupled reversed electron transfer in Klebsiella pneumoniae

Citrate is fermented by Klebsiella pneumoniae to 2 acetate, 0.5 formate and 1.2 CO2. The formation of less than 1 formate and greater than 1 CO2 per citrate can be accounted for by the oxidation of formate to CO2 in order to provide reducing equivalents for the assimilation of citrate into cell carbon. A membrane-bound electron transport chain is apparently involved in NADH synthesis by these cells. The electrons from formate oxidation to CO2 are used to reduce ubiquinone to ubiquinol by membrane-bound formate dehydrogenase and ubiquinol further delivers its electrons to NAD+, if this endergonic reaction is powered by delta mu Na+. The endogenous NADH level of K. pneumoniae cells thus increased in the presence of formate in response to a delta pNa+ greater than -100 mV. NADH formation was completely abolished in the presence of oxygen or after addition of hydroxyquinoline-N-oxide, a specific inhibitor of the Na(+)-translocating NADH:ubiquinone oxidoreductase. The increase of endogenous NADH was dependent on the delta pNa+ applied to the cells. Inverted membrane vesicles of K. pneumoniae catalysed the reduction of NAD+ to NADH with formate as electron donor after application of delta mu Na+ of about 120 mV consisting of delta pNa+ of 60 mV and delta psi of the same magnitude. Neither the delta pNa+ nor the delta psi of this size alone was sufficient to drive the endergonic reaction. Strictly anaerobic conditions were required for NADH formation and hydroxyquinoline-N-oxide completely inactivated the reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+ translocation by the NADH:ubiquinone oxidoreductase (complex I) from Klebsiella pneumoniae

Complex I is the site for electrons entering the respiratory chain and therefore of prime importance for the conservation of cell energy. It is generally accepted that the complex I-catalysed oxidation of NADH by ubiquinone is coupled specifically to proton translocation across the membrane. In variance to this view, we show here that complex I of Klebsiella pneumoniae operates as a primary Na+ pump. Membranes from Klebsiella pneumoniae catalysed Na+-stimulated electron transfer from NADH or deaminoNADH to ubiquinone-1 (0.1-0.2 micromol min-1 mg-1). Upon NADH or deaminoNADH oxidation, Na+ ions were transported into the lumen of inverted membrane vesicles. Rate and extent of Na+ transport were significantly enhanced by the uncoupler carbonylcyanide-m-chlorophenylhydrazone (CCCP) to values of approximately 0.2 micromol min-1 mg-1 protein. This characterizes the responsible enzyme as a primary Na+ pump. The uptake of sodium ions was severely inhibited by the complex I-specific inhibitor rotenone with deaminoNADH or NADH as substrate. N-terminal amino acid sequence analyses of the partially purified Na+-stimulated NADH:ubiquinone oxidoreductase from K. pneumoniae revealed that two polypeptides were highly similar to the NuoF and NuoG subunits from the H+-translocating NADH:ubiquinone oxidoreductases from enterobacteria.