DsrL mediates electron transfer between NADH and rDsrAB in Allochromatium vinosum.

Dissimilatory sulphite reductase DsrAB occurs in sulphate/sulphite-reducing prokaryotes, in sulphur disproportionators and also in sulphur oxidizers, where it functions in reverse. Predictions of physiological traits in metagenomic studies relying on the presence of dsrAB, other dsr genes or combinations thereof suffer from the lack of information on crucial Dsr proteins. The iron-sulphur flavoprotein DsrL is an example of this group. It has a documented essential function during sulphur oxidation and was recently also found in some metagenomes of probable sulphate and sulphite reducers. Here, we show that DsrL and reverse acting rDsrAB can form a complex and are copurified from the phototrophic sulphur oxidizer Allochromatium vinosum. Recombinant DsrL exhibits NAD(P)H:acceptor oxidoreductase activity with a strong preference for NADH over NADPH. In vitro, the rDsrABL complex effectively catalyses NADH-dependent sulphite reduction, which is strongly enhanced by the sulphur-binding protein DsrC. Our work reveals NAD+ as suitable in vivo electron acceptor for sulphur oxidation in organisms operating the rDsr pathway and points to reduced nicotinamide adenine dinucleotides as electron donors for sulphite reduction in sulphate/sulphite-reducing prokaryotes that contain DsrL. In addition, dsrL cannot be used as a marker distinguishing sulphate/sulphite reducers and sulphur oxidizers in metagenomic studies without further analysis.

Electronic structure of the lowest triplet state of flavin mononucleotide.

The electronic structure of flavin mononucleotide (FMN), an organic cofactor that plays a role in many important enzymatic reactions, has been investigated by electron paramagnetic resonance (EPR) spectroscopy, optical spectroscopy, and quantum chemistry. In particular, the triplet state of FMN, which is paramagnetic (total spin S = 1), allows an investigation of the zero field splitting parameters D and E, which are directly related to the two singly occupied molecular orbitals. Triplet EPR spectra and optical absorption spectra at different pH values in combination with time dependent density functional theory (TDDFT) reveal that the highest occupied orbital (HOMO) and lowest unoccupied orbital (LUMO) of FMN are largely unaffected by changes in the protonation state of FMN. Rather, the orbital structure of the lower lying doubly occupied orbitals changes dramatically. Additional EPR experiments have been carried out in the presence of AgNO(3), which allows the formation of an Ag-FMN triplet state with different zero field splitting parameters and population and depopulation rates. Addition of AgNO(3) only induces small changes in the optical spectrum, indicating that the Ag(+) ion only contributes to the zero field splitting by second order spin-orbit coupling and leaves the orbital structure unaffected. By a combination of the three employed methods, the observed bands in the UV/vis spectra of FMN at different pH values are assigned to electronic transitions.