Spontaneous activation of [FeFe]-hydrogenases by an inorganic [2Fe] active site mimic

Abstract

Hydrogenases catalyze the formation of hydrogen. The cofactor ('H-cluster') of [FeFe]-hydrogenases consists of a [4Fe-4S] cluster bridged to a unique [2Fe] subcluster whose biosynthesis in vivo requires hydrogenase-specific maturases. Here we show that a chemical mimic of the [2Fe] subcluster can reconstitute apo-hydrogenase to full activity, independent of helper proteins. The assembled H-cluster is virtually indistinguishable from the native cofactor. This procedure will be a powerful tool for developing new artificial H₂-producing catalysts.

Figure 1. The H-cluster is assembled from a regular [4Fe-4S]-cluster and a unique [2Fe]-subcluster

The synthetic [2Fe]-subcluster ([2Fe]^MIM) differs from the binuclear subsite of the H-cluster by an additional CO-group. Models of the H- and the [4Fe-4S]-clusters were generated in PyMOL on the basis of PDB structures 3C8Y and 3LX4, respectively. [2Fe]^MIM was also modeled in PyMOL according to information given in . Some amino acids were deleted to provide an unobstructed view of the clusters. The metal clusters are in the ball-and-stick representation, the coordinating cysteines are shown as sticks. Orange: iron, yellow: sulfur, gray: carbon, blue: nitrogen, red: oxygen.

Figure 2. Various types of HYDA enzymes can be spontaneously activated by [2Fe]^MIM and behave like the natural enzymes

a, Specific H₂ evolution activities of C. reinhardtii HYDA1 after combination of apoHYDA1 with [2Fe]^MIM alone or [2Fe]^MIM-loaded HYDF. Molar ratios are indicated on the x-axis. b, HYDA1^MIM reached the same specific activity as HYDA1 heterologously produced in C. acetobutylicum while neither [2Fe]^MIM nor apoHYDA1 showed any activities. [2Fe]^MIM also activated apoHYDA enzymes of M. elsdenii (HYDA Me MIM) and C. pasteurianum (CpI MIM). We measured H₂ evolution using reduced methyl viologen as electron donor. c, H₂ production was also achieved in reconstitution assays including pyruvate, acetylCoA, pyruvate:ferredoxin oxidoreductase PFR1 and ferredoxin PETF in the dark (pyruvate-driven) or with photosystem 1 (PS1), PETF and the necessary PS1 reducing agents in the light (light-driven). a – c, All values shown are mean values from at least four independent experiments. Error bars indicate the standard deviation.

Figure 3. The chemically reconstituted H-cluster is virtually indistinguishable from the native form

a, EPR-spectra of apoHYDA1,HYDA1^MIM in the CO-inhibited H_ox-CO state and as isolated HYDA1^MIM comprising all EPR active states. b, FTIR spectra of [2Fe]^MIM in solution,of CO-treated HYDA1^MIM and as isolated HYDA1^MIM . EPR simulations for each state are shown^, in the supplementary results (Supplementary Fig. 2a). The g-values are indicated above the spectra. The assignments of the FTIR signals to the different redox states are collected in Supplementary Fig. 2b. All spectra were recorded on two independently prepared samples. One representative result is shown.