Transforming an oxygen-tolerant [NiFe] uptake hydrogenase into a proficient, reversible hydrogen producer
Many hydrogenases are highly electroactive when attached to an electrode, and most exhibit reversible 2H+/H2 electrocatalysis, i.e. only a minuscule overpotential is required to drive the reaction in either direction. A notable exception is an important class of membrane-bound O2-tolerant [NiFe] hydrogenases that appear only to catalyse H2 oxidation (the uptake reaction), at a substantial overpotential and with little activity for H2 production, yet possess an active site that is structurally very similar to that of standard, reversible [NiFe] hydrogenases (Volbeda et al., Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 5305–5310). In a discovery providing important insight into this puzzle, we show that the O2-tolerant [NiFe] hydrogenase (Hyd-1) from E. coli converts into a reversible electrocatalyst as the pH is lowered from 8 to 3 and becomes an efficient H2 producer below pH 4. The transformation to a reversible electrocatalyst is not due, trivially, to the higher substrate (H+aq) availability at low pH but to a large shift in the enzyme's catalytic bias. Systematic investigations provide compelling evidence that the factor controlling this behaviour is the distal [4Fe–4S] cluster, a spectroscopically elusive site that provides the natural entry point for electrons into the enzyme. In E. coli cells, Hyd-1 is located in the periplasmic (extracytoplasmic) compartment and thus, being exposed to the pH extremes of the gastrointestinal tract or the external environment, is a potential catalyst for H2 production by these bacteria. In a wider context, the observation and proposal are highly relevant for biohydrogen production and catalysis.