NADP + or CO 2 reduction by frhAGB -encoded hydrogenase through interaction with formate dehydrogenase 3 in the hyperthermophilic archaeon Thermococcus onnurineus NA1

Abstract

ABSTRACT It has been reported that the frhAGB -encoded hydrogenase from Thermococcus onnurineus NA1 is homologous to the F 420 -reducing hydrogenase from methanogens and can reduce thioredoxin reductase (TrxR) via direct electron transfer from H 2 oxidation. In this study, to find other interaction targets of frhAGB -encoded hydrogenase, we searched for structural homologs of TrxR using the Position-Specific Iterative Basic Local Alignment Search Tool (PSI-BLAST) protein database search program and AlphaFold Protein Structure Database. Fdh3B (TON_0542), a subunit of the formate dehydrogenase 3 (Fdh3), showed the most similar structure to TrxR in its domains. The interaction potential of Fdh3B with frhAGB -encoded hydrogenase was demonstrated by measuring H 2 -dependent NADP + reduction by a mixture of purified proteins. Similarly, the H 2 -dependent NADP + -reducing activity of Fdh3 whole complex and two other TrxR homologs encoded by TON_0702 and TON_1376 was determined. It was also demonstrated that the Fdh3 complex could reduce CO 2 to formate in the presence of the frhAGB -encoded hydrogenase and H 2 , as shown for the H 2 -dependent CO 2 reductase (HDCR) of acetogen. The in vivo contribution of the frhAGB -encoded hydrogenase and Fdh3 to H 2 -dependent CO 2 reduction was investigated using a resting cell assay, and formate production was significantly decreased in fdh3A or frhA deletion mutants. In conclusion, the frhAGB -encoded hydrogenase was identified to directly transfer electrons to Fdh3 without an electron carrier, similar to the mechanism observed for TrxR. These results expand our understanding of the functional role of the frhAGB -encoded hydrogenase in non-methanogenic Thermococcus species. IMPORTANCE The strategy using structural homology with the help of structure prediction by AlphaFold was very successful in finding potential targets for the frhAGB -encoded hydrogenase of Thermococcus onnurineus NA1. The finding that the hydrogenase can interact with FdhB to reduce the cofactor NAD(P) + is significant in that the enzyme can function to supply reducing equivalents, just as F 420 -reducing hydrogenases in methanogens use coenzyme F 420 as an electron carrier. Additionally, it was identified that T. onnurineus NA1 could produce formate from H 2 and CO 2 by the concerted action of frhAGB -encoded hydrogenase and formate dehydrogenase Fdh3.