Structure-activity relationship study shows versatility in substrate specificity for heterodisulfide reductase in methanogenic archaea.

Hydrogenotrophic methanogens are anaerobic archaea that convert carbon dioxide and hydrogen into methane. Central to this process is the heterodisulfide reductase (Hdr), which catalyzes the reduction of the heterodisulfide made of coenzyme M and coenzyme B. In vivo, Hdr functions in association with electron-donating modules such as the [NiFe]-hydrogenase (HdrMvh), which supplies reducing equivalents derived from hydrogen oxidation. Here, we isolate the catalytic properties of Hdr by evaluating substrate turnover independently of its native electron-donating modules using an artificial electron donor system. The molecular features governing substrate recognition and turnover by Hdr were investigated through the design and synthesis of 18 non-endogenous analogs of the native heterodisulfide substrate (compounds 2a-r). We show that several non-endogenous disulfides can serve as alternative substrates for Hdr, including analogs with variation in the coenzyme B-derived amino acid moiety (O-phosphono-Ser, L-Asp, L-Glu, and 2-aminoadipic acid), and that the coenzyme M sulfonate can be replaced by a carboxylate group. In contrast, modifications that disrupt charge balance, chain length, or stereochemical compatibility result in loss of substrate activity and, in some cases, lead to inhibitory behavior, underscoring the narrow constraints required for productive catalysis.