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Influence of the [4Fe-4S] cluster coordinating cysteines on active site maturation and catalytic properties of C. reinhardtii [FeFe]-hydrogenase.

ABSTRACT: [FeFe]-Hydrogenases catalyze the evolution and oxidation of hydrogen using a characteristic cofactor, termed the H-cluster. This comprises an all cysteine coordinated [4Fe-4S] cluster and a unique [2Fe] moiety, coupled together via a single cysteine. The coordination of the [4Fe-4S] cluster in HydA1 from Chlamydomonas reinhardtii was altered by single exchange of each cysteine (C115, C170, C362, and C366) with alanine, aspartate, or serine using site-directed mutagenesis. In contrast to cysteine 115, the other three cysteines were found to be dispensable for stable [4Fe-4S] cluster incorporation based on iron determination, UV/vis spectroscopy and electron paramagnetic resonance. However, the presence of a preformed [4Fe-4S] cluster alone does not guarantee stable incorporation of the [2Fe] cluster. Only variants C170D, C170S, C362D, and C362S showed characteristic signals for an inserted [2Fe] cluster in Fourier-transform infrared spectroscopy. Hydrogen evolution and oxidation were observed for these variants in solution based assays and protein-film electrochemistry. Catalytic activity was lowered for all variants and the ability to operate in either direction was also influenced.

SUBMITTER: Kertess L

PROVIDER: S-EPMC5855289 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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A [4Fe-4S]-Fe(CO)(CN)-L-cysteine intermediate is the first organometallic precursor in [FeFe] hydrogenase H-cluster bioassembly.

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Project description:The [FeFe] hydrogenases HydA1 and HydA2 in the green alga Chlamydomonas reinhardtii catalyze the final reaction in a remarkable metabolic pathway allowing this photosynthetic organism to produce H(2) from water in the chloroplast. A [2Fe-2S] ferredoxin is a critical branch point in electron flow from Photosystem I toward a variety of metabolic fates, including proton reduction by hydrogenases. To better understand the binding determinants involved in ferredoxin:hydrogenase interactions, we have modeled Chlamydomonas PetF1 and HydA2 based on amino-acid sequence homology, and produced two promising electron-transfer model complexes by computational docking. To characterize these models, quantitative free energy calculations at atomic resolution were carried out, and detailed analysis of the interprotein interactions undertaken. The protein complex model we propose for ferredoxin:HydA2 interaction is energetically favored over the alternative candidate by 20 kcal/mol. This proposed model of the electron-transfer complex between PetF1 and HydA2 permits a more detailed view of the molecular events leading up to H(2) evolution, and suggests potential mutagenic strategies to modulate electron flow to HydA2.

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