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

Formate hydrogenlyases are metalloenzymes that can either produce molecular hydrogen gas or use H2 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 H2 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 H2 and CO2. 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.