Anaerobic bacteria ferment glutamate via two different pathways to ammonia, carbon dioxide, acetate, butyrate and molecular hydrogen. The coenzyme B₁₂-dependent pathway in Clostridium tetanomorphum via 3-methylaspartate involves pyruvate:ferredoxin oxidoreductase and a novel enzyme, a membrane-bound NADH:ferredoxin oxidoreductase. The flavin- and iron-sulfur-containing enzyme probably uses the energy difference between reduced ferredoxin and NADH to generate an electrochemical Na⁺ gradient, which drives transport processes. The other pathway via 2-hydroxyglutarate in Acidaminococcus fermentans and Fusobacterium nucleatum involves glutaconyl-CoA decarboxylase, which uses the free energy of decarboxylation to generate also an electrochemical Na⁺ gradient. In the latter two organisms, similar membrane-bound NADH:ferredoxin oxidoreductases have been characterized. We propose that in the hydroxyglutarate pathway these oxidoreductases work in the reverse direction, whereby the reduction of ferredoxin by NADH is driven by the Na⁺ gradient. The reduced ferredoxin is required for hydrogen production and the activation of radical enzymes. Further examples show that reduced ferredoxin is an agent, whose reducing energy is about 1 ATP ‘richer’ than that of NADH.

The respiratory NADH:quinone oxidoreductase (complex I) (NDH-1) is a multisubunit enzyme that translocates protons (or in some cases Na ؉ ) across energy-conserving membranes from bacteria or mitochondria. We studied the reaction of the Na ؉ -translocating complex I from the enterobacterium Klebsiella pneumoniae with N,N-dicyclohexylcarbodiimide (DCCD), with the aim of identifying a subunit critical for Na ؉ binding. At low Na ؉ concentrations (0.6 mM), DCCD inhibited both quinone reduction and Na ؉ transport by NDH-1 concurrent with the covalent modification of a 30-kDa polypeptide. In the presence of 50 mM Na ؉ , NDH-1 was protected from inhibition by DCCD, and the modification of the 30-kDa polypeptide with [ 14 C]DCCD was prevented, indicating that Na ؉ and DCCD competed for the binding to a critical carboxyl group in NDH-1. The 30-kDa polypeptide was assigned to NuoH, the homologue of the ND1 subunit from mitochondrial complex I. It is proposed that Na ؉ binds to the NuoH subunit during NADH-driven Na ؉ transport by NDH-1.

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