Pectobacterium atrosepticum SCRI1043 is a phytopathogenic Gram-negative enterobacterium. Genomic analysis has identified that genes required for both respiration and fermentation are expressed under anaerobic conditions. One set of anaerobically expressed genes is predicted to encode an important but poorly understood membrane-bound enzyme termed formate hydrogenlyase-2 (FHL-2), which has fascinating evolutionary links to the mitochondrial NADH dehydrogenase (Complex I). In this work, molecular genetic and biochemical approaches were taken to establish that FHL-2 is fully functional in P. atrosepticum and is the major source of molecular hydrogen gas generated by this bacterium. The FHL-2 complex was shown to comprise a rare example of an active [NiFe]-hydrogenase-4 (Hyd-4) isoenzyme, itself linked to an unusual selenium-free formate dehydrogenase in the final complex. In addition, further genetic dissection of the genes encoding the predicted membrane arm of FHL-2 established surprisingly that the majority of genes encoding this domain are not required for physiological hydrogen production activity. Overall, this study presents P. atrosepticum as a new model bacterial system for understanding anaerobic formate and hydrogen metabolism in general, and FHL-2 function and structure in particular. © 2019 John Wiley & Sons Ltd.