Monitoring H-cluster assembly using a semi-synthetic HydF proteinElectronic supplementary information (ESI) available. See DOI: 10.1039/c8dt04294b

• Main Authors: Németh, Brigitta, Esmieu, Charlène, Redman, Holly J, Berggren, Gustav
• Format: Article
• Language: English
• ISSN: 1477-9226; 1477-9234
• DOI: 10.1039/c8dt04294b

The [FeFe] hydrogenase enzyme interconverts protons and molecular hydrogen with remarkable efficiency. The reaction is catalysed by a unique metallo-cofactor denoted as the H-cluster containing an organometallic dinuclear Fe component, the [2Fe] subsite. The HydF protein delivers a precursor of the [2Fe] subsite to the apo-[FeFe] hydrogenase, thus completing the H-cluster and activating the enzyme. Herein we generate a semi-synthetic form of HydF by loading it with a synthetic low valent dinuclear Fe complex. We show that this semi-synthetic protein is practically indistinguishable from the native protein, and utilize this form of HydF to explore the mechanism of H-cluster assembly. More specifically, we show that transfer of the precatalyst from HydF to the hydrogenase enzyme results in the release of CO, underscoring that the pre-catalyst is a four CO species when bound to HydF. Moreover, we propose that an electron transfer reaction occurs during H-cluster assembly, resulting in an oxidation of the [2Fe] subsite with concomitant reduction of the [4Fe4S] cluster present on the HydF protein. An artificial enzyme, incorporating an organometallic iron complex, is used to probe the activation mechanism of [FeFe] hydrogenase.