Abstract
rnf genes are widespread in bacteria and biochemical and genetic data are in line with the hypothesis that they encode a membrane-bound enzyme that oxidizes reduced ferredoxin and reduces NAD and vice versa, coupled to ion transport across the cytoplasmic membrane. The Rnf complex is of critical importance in many bacteria for energy conservation but also for reverse electron transport to drive ferredoxin reduction. However, the enzyme has never been purified and thus, ion transport could not be demonstrated yet. Here, we have purified the Rnf complex from the anaerobic, fermenting thermophilic bacterium Thermotoga maritima and show that is a primary Na+ pump. These studies provide the proof that the Rnf complex is indeed an ion (Na+) translocating, respiratory enzyme. Together with a Na+-F1FO ATP synthase it builds a simple, two-limb respiratory chain in T. maritima. The physiological role of electron transport phosphorylation in a fermenting bacterium is discussed.
Highlights
Rnf genes are widespread in bacteria and biochemical and genetic data are in line with the hypothesis that they encode a membrane-bound enzyme that oxidizes reduced ferredoxin and reduces NAD and vice versa, coupled to ion transport across the cytoplasmic membrane
The activity was enriched from membranes of A. woodii and the preparation contained, amongst others, proteins with similarity to proteins encoded by the rnf genes[19]
We describe here a procedure to purify the Rnf complex from a thermophilic bacterium, Thermotoga maritima, in complex with the ATP synthase
Summary
Rnf genes are widespread in bacteria and biochemical and genetic data are in line with the hypothesis that they encode a membrane-bound enzyme that oxidizes reduced ferredoxin and reduces NAD and vice versa, coupled to ion transport across the cytoplasmic membrane. This raised the question whether the Rnf complex from T. maritima would accept ferredoxin from Clostridium pasteurianum, which is generally used as electron donor to measure Fdred:NAD-oxidoreductase activity. The Fd2−: NAD+-oxidoreductase activity at membranes of T. maritima was 97 mU/mg, measured at 60 °C (Supplementary Fig. 1).
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