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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 f...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2008
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| Zusammenfassung: | 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. |
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| Umfang: | 106 Seiten |
| DOI: | 10.17192/z2009.0712 |