Electron Donor Systems to Facilitate Development of Assays for Two Flavoproteins Involved in Tetrahydromethanopterin Biosynthesis

Methane production by archaea depends on tetrahydromethanopterin (H4MPT), a pterin-containing cofactor that carries one-carbon units. Two redox reactions within the nine steps of H4MPT side chain biosynthesis have been hypothesized. Biochemical assays have demonstrated that the archaeal iron-sulfur flavoprotein dihydromethanopterin reductase X (DmrX or MM1854) catalyzes the final reaction of the pathway, the reduction of dihydromethanopterin to H4MPT, using dithiothreitol (DTT) as an artificial electron donor. The crystal structure of DmrB, a bacterial DmrX homolog that lacks iron-sulfur clusters, has led to a proposed ping-pong mechanism of electron transfer between FMNH2 and the FMN prosthetic group of DmrB. However, an enzymatic assay to test the hypothetical DmrB mechanism is lacking because a suitable electron donor has not previously been identified. Furthermore, a second uncharacterized archaeal flavoprotein (MM1853) has been hypothesized to function in H4MPT side chain biosynthesis. In this work, to facilitate the development of assays to elucidate the functions of DmrB and MM1853, we tested a variety of electron donors, including dithiothreitol, ferredoxin, and a system consisting of NADH and an NADH-dependent flavin-reducing enzyme (Fre). Reduction of the DmrB prosthetic group (FMN) was measured as a decrease in absorbance at 460 nm. NADPH, NADH, and DTT were unable to reduce DmrB. However, NADH/Fre was able to reduce DmrB within 70 min (initial rate of 1.3 μM/min), providing the basis for a future DmrB activity assay. Carbon monoxide (CO)/CO dehydrogenase/ferredoxin reduced DmrB more rapidly within 6 min. Both electron transfer systems reduced a second flavin-containing archaeal protein MM1853, which is predicted to catalyze the third step of H4MPT biosynthesis. While NADH and NADPH were incapable of directly reducing the FMN cofactor of MM1853, DTT or NADH/Fre could eliminate the FMN peaks. These results establish the basis for new oxidoreductase assays that will facilitate testing several proposed DmrB mechanisms and defining the specific function of MM1853 in methanogen cofactor biosynthesis.