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Characterization of a nifâregulated flavoprotein (FprA) from Rhodobacterâcapsulatus: Redox properties and molecular interaction with a [2Feâ2S] ferredoxin
- Jouanneau, Yves, Meyer, Christine, Asso, Marcel, Guigliarelli, Bruno, Willison, John C.
- European journal of biochemistry 2000 v.267 no.3 pp. 780-787
- Escherichia coli, Rhodobacter capsulatus, ferredoxins, flavoproteins, hydroquinone, mutants, nitrogen fixation, nitrogenase, pH, redox potential, structural genes, thermodynamics, titration
- A flavoprotein from Rhodobacter capsulatus was purified as a recombinant (His)6âtag fusion from an Escherichiaâcoli clone overâexpressing the fprA structural gene. The FprA protein is a homodimer containing one molecule of FMN per 48âkDa monomer. Reduction of the flavoprotein by dithionite showed biphasic kinetics, starting with a fast step of semiquinone (SQ) formation, and followed by a slow reduction of the SQ. This SQ was in the anionic form as shown by EPR and optical spectroscopies. Spectrophotometric titration gave a midpoint redox potential for the oxidized/SQ couple of Em1â=â+20âmV (pHâ8.0), whereas the SQ/hydroquinone couple could not be titrated due to the thermodynamic instability of SQ associated with its slow reduction process. The inability to detect the intermediate form, SQ, upon oxidative titration confirmed this instability and led to an estimate of Em2âââEm1 of >â80âmV. The reduction of SQ by dithionite was significantly accelerated when the [2Feâ2S] ferredoxin FdIV was used as redox mediator. The midpoint redox potential of this ferredoxin was determined to be â275âÂ±â2âmV at pHâ7.5, consistent with FdIV serving as electron donor to FprA in vivo. FdIV and FprA were found to crossâreact when incubated together with the 1âethylâ3â(3âdimethylaminopropyl)carbodiimide, giving a covalent complex with an Mr of ââ60â000. Formation of this complex was unaffected by the redox states of the two proteins. Other [2Feâ2S] ferredoxins, including FdV and FdVI from R.âcapsulatus, were ineffective as electron carriers to FprA, and crossâreacted poorly with the flavoprotein. The possible function of FprA with regard to nitrogen fixation was investigated using an fprAâdeleted mutant. Although nitrogenase activity was significantly reduced in the mutant compared with the wildâtype strain, nitrogen fixation was apparently unaffected by the fprA deletion even under iron limitation or microaerobic conditions.