Characterization of a nif-regulated flavoprotein (FprA) from Rhodobacter capsulatus - Redox properties and molecular interaction with a [2Fe-2S] ferredoxin

A flavoprotein from Rhodobacter capsulatus was purified as a recombinant (H is)(6)-tag fusion from an Escherichia coli clone over-expressing the fprA s tructural gene. The FprA protein is a homodimer containing one molecule of FMN per 48-kDa 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 g ave a midpoint redox potential for the oxidized/SQ couple of E-m1 = +20 mV (pH 8.0), whereas the SQ/hydroquinone couple could not be titrated due to t he thermodynamic instability of SQ associated with its slow reduction proce ss. The inability to detect the intermediate form, SCE, upon oxidative titr ation confirmed this instability and led to an estimate of E-m2 - E-m1 of > 80 mV, The reduction of SQ by dithionite was significantly accelerated whe n the [2Fe-2S] ferredoxin FdIV was used as redox mediator The midpoint redo x potential of this ferredoxin was determined to be -275 +/- 2 mV at pH 7.5 , consistent with FdIV serving as electron donor to FprA in vivo, FdIV and FprA were found to cross-react when incubated together with the 1-ethyl-3-( 3-dimethylaminopropyl)carbodiimide, giving a covalent complex with an M-r o f approximate to 60 000. Formation of this complex was unaffected by the re dox states of the two proteins. Other [2Fe-2S] ferredoxins, including FdV a nd FdVI from R. capsulatus, were ineffective as electron carriers to FprA, and cross-reacted poorly with the flavoprotein, The possible function of Fp rA with regard to nitrogen fixation was investigated using an fprA-deleted mutant. Although nitrogenase activity was significantly reduced in the muta nt compared with the wild-type strain, nitrogen fixation was apparently una ffected by the fprA deletion even under iron limitation or microaerobic con ditions.