Genetic dissection of formate hydrogenlyase-2 function: a metalloenzyme involved in bacterial hydrogen production

Abstract Formate hydrogenlyases are metalloenzymes that can either produce molecular hydrogen gas or use H 2 to convert carbon dioxide to formic acid, thus potentially contributing doubly to a sustainable energy future. The structure of formate hydrogenlyase reveals a membrane-bound redox enzyme that shares a common ancestor with the mitochondrial complex I (NADH dehydrogenase). As such, formate hydrogenlyase falls into a category of so‐called ‘complex‐I‐like’ enzymes, that are found in bacteria, archaea and eukaryotic organelles. They share a common core structure of a membrane arm (most likely involved in proton or ion translocation) and a peripheral arm containing metal cofactors and involved in electron transfer. In this work, we clone a gene cluster from Pectobacterium atrosepticum encoding formate hydrogenlyase-2 (FHL-2). A bank of Escherichia coli host strains, themselves devoid of various combinations of native formate hydrogenlyase genes, are employed to characterise FHL-2. We demonstrate that P. atrosepticum FHL-2 is active in an E. coli host in that it can generate H 2 under fermentative growth conditions. Unlike native E. coli formate hydrogenlyase-1 (FHL-1), recombinant P. atrosepticum FHL-2 cannot perform the reverse reaction and generate formic acid from H 2 and CO 2 . By testing different combinations of genes by taking an in vivo cross-complementation approach we conclude that the extended membrane arm exhibited by FHL-2 is a major factor in controlling directionality of the enzyme.