A unique way of energy conservation in glutamate fermenting clostridia

Genetic analysis revealed that Rhodobacter capsulatus contains six rnfABCDEG-genes that are responsible for the electron flow in nitrogen fixation (rnf = Rhodobacter nitrogen fixation). Homolgous genes have been detected in Clostridium tetani. In this work, a membrane complex has been purified from the related Clostridium tetanomorphum that catalyses the reduction of NAD + (E°' = −320 mV) with ferredoxin (E°' ≤ −420 mV). The difference in the redox potential of ≥ 100 mV could be useful for additional energy conservation in the fermentation of glutamate to ammonia, CO 2 , acetate, butyrate, and H 2 . The complex consists of six subunits (RnfABCDEG), of which four N-termini (RnfCDEG) could be sequenced. The sequences are 60-80% identical to the deduced sequences of the Rnf-subunits from C. tetani. The rnf operon has been completely sequenced and aligned with the sequences of C. tetani. The complex contains both non-covalently bound flavin as well as covalently bound flavin. The non-covalently bound flavin was identified as FMN and riboflavin in 1:1 stochiometric ratio, each 0.3 mol/mol Rnf complex (180 kDa). The subunits RnfG and RnfD contain covalently bound flavin linked via phosphodiester bond. The iron was determined as 25±1 mol per Rnf complex. Usually, Rnf activity was measured with NADH and ferricyanide at 420 nm. In order to measure NAD + reduction with reduced ferredoxin catalysed by Rnf complex, the ferredoxin was purified from C. tetanomorphum and reduced by Ti(III)citrate at pH 7.0. High Rnf activities were observed in the membrane preparations of Clostridium aminobutyricum, Clostridium pascui and Clostridium propionicum. Thus, additional energy conservation can be explained in these bacteria. However Rnf activity was absent in Eubacterium barkeri, a nicotinate fermenting bacteria. The soluble butyryl-CoA-dehydrogenase/electron transferring flavoprotein (Bcd/Etf) complex was purified from C. pascui as well as from C. tetanomorphum. The N- terminal sequences of the three subunits (αβγ) showed high identities with the deduced sequences of C. tetani. The Bcd/Etf complex purified from C. tetanomorphum was shown to catalyze the endergonic reduction of ferredoxin with NADH coupled to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E°' = -10 mV) with NADH. The12 reduced ferredoxin could be used for H 2 production catalysed by a hydrogenase or probably used for additional energy conservation via Rnf (about 0.3 mol ATP/ mol glutamate). Experiments with [2,4,4- 2 H] glutamate and detection of citramalate-lyase activity showed that C. pascui and C. tetanomorphum ferment glutamate via the methylaspartate pathway