The exchange activities of [Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) from methanogenic archaea in comparison with the exchange activities of [FeFe] and [NiFe] hydrogenases

[Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) catalyzes the reversible reduction of methenyltetrahydromethanopterin (methenyl-H(4)MPT(+)) with H(2) to methylene-H(4)MPT, a reaction involved in methanogenesis from H(2) and CO(2) in many methanogenic archaea. The enzyme harbors an iron-containing cofactor, in which a low-spin iron is complexed by a pyridone, two CO and a cysteine sulfur. [Fe] hydrogenase is thus similar to [NiFe] and [FeFe] hydrogenases, in which a low-spin iron carbonyl complex, albeit in a dinuclear metal center, is also involved in H(2) activation. Like the [NiFe] and [FeFe] hydrogenases, [Fe] hydrogenase catalyzes an active exchange of H(2) with protons of water; however, this activity is dependent on the presence of the hydride-accepting methenyl-H(4)MPT(+). In its absence the exchange activity is only 0.01% of that in its presence. The residual activity has been attributed to the presence of traces of methenyl-H(4)MPT(+) in the enzyme preparations, but it could also reflect a weak binding of H(2) to the iron in the absence of methenyl-H(4)MPT(+). To test this we reinvestigated the exchange activity with [Fe] hydrogenase reconstituted from apoprotein heterologously produced in Escherichia coli and highly purified iron-containing cofactor and found that in the absence of added methenyl-H(4)MPT(+) the exchange activity was below the detection limit of the tritium method employed (0.1 nmol min(−1) mg(−1)). The finding reiterates that for H(2) activation by [Fe] hydrogenase the presence of the hydride-accepting methenyl-H(4)MPT(+) is essentially required. This differentiates [Fe] hydrogenase from [FeFe] and [NiFe] hydrogenases, which actively catalyze H(2)/H(2)O exchange in the absence of exogenous electron acceptors.