Compartmental Maturation Of [FeFe]-hydrogenase HydA1 In Chlamydomonas Reinhardtii

The unicellular microalgae Chlamydomonas reinhardtii is one of the best-studied hydrogen-producing microalgae.It contains two nuclear-encoded hydrogenase isoforms,HydA1 and HydA2,both of which are capable of enrering the chloroplast and reversely catalyzing the proton reduction to generate hydrogen via the photosynthesis electron transport chain.The extensive studies mainly focus on the HydAl,that shows much higher enzymatic activity than HydA2 and catalyzes about 75%hydrogen production in the light.The structure of Chlamydomonas reinhardtii hydrogense is the simplest among[FeFe]hydrogenases,with its active site,namely H-cluster composed of a classic[4Fe4S]linked with a unique[2Fe2S]sub-cluster through the cysteine residue.It is confirmed that the[4Fe4S]cluster is incorporated onto the protein first by the housekeeping FeS assembly machinery followed by the[2Fe2S]sub-cluster incorporation by the specific maturases HydE,HydF and HydG.There are three[4Fe4S]assembly machineries in C.reinhardtii,the ISC(iron sulfur cluster)in the mitochondria,CIA(cytosolic iron sulfur assembly)in the cytosol and SUF(sulfur utilization factor)in the chloroplast.Like HydAl,the maturases are also encoded in the nuclear genome and found functioning in the chloroplast.To investigate the HydAl maturation process in C.reinhardtii,the hydAl genes which include the full-length and the chloroplast transit peptide(cpTP)removed hydA1 cDNA(TPphts-chydAl and TPminus-chydA1)and as well as the full-length and the cpTP removed genomic hydAl(TPphus-ghydA1 and TPminus-ghydA1)are fused with Hsp70A/rbcs2 or HydAl native promoter and transformed into a hydrogenase deficient C.reinhardtii hydA1-1hydA2-1.But the expression of HydAl cDNA failed with an unknown reason that needs to be explored further.The homologous expression system of hydrogenase HydAl in the mutant hydA11hydA2-1has been set up and parallelly the corresponding screening and analysis methods,such as antibiotic concentration determination,colony PCR,enzymatic activity assay,hydrogenase purification from microalgae,western-blot assay,in vitro[2Fe2S]maturation and[4Fe4S]reconstitution.Four kinds of recombinant transformants have been screened out,Hsp70A/rbcs2-TPplus-ghydA1-aph7(Hsp+),Hsp 70A/rbcs2-TPminus-ghydA1-aph7(Hsp-),HydA1 promoter-TPplus-ghydA1-aph7(HA+),HydA1 promoter-TPminus-ghydAl-aph7(HA-).Two transformants of Hsp70A/rbcs2-TPplus-ghydAl-aph7,Hsp?1 and Hsp+2 showed even higher in vitro hydrogenase activity,34.3%and 68.5%higher than the wildtype CC124,indicating the high potential for further optimization and industrial application.The HydAl protein could be purified with high activity from Hsp?1 and Hsp+2 after induced via the classic sulfur deficiency or anaerobiosis adaption.In contrast,all the transformants without the cpTP exhibited extremely low in vitro hydrogenase activity and the corresponding purified enzyme displayed very low activity as well.However,the activity could be slightly improved after in vitro maturation by adding the[2Fe]MIM,the chemical mimic of[2Fe2S]sub-cluster,which could activate the apo-HydAl that lacks the[2Fe2S],while it is significantly activated after the in vitro[4Fe4S]reconstitution and[2Fe]MIM addition.This indicated that the cytoplasm-trapped HydAl couldn't be equipped with FeS cluster,which revealed that the FeS cluster can't be assembled before entering the choloroplast.Utilizing the same expression frame,the green fluorescent protein(GFP)fused with HydA1 chloroplast transit peptide(cpTP)at the 5' terminus was successfully expressed in the mutant C.reinhardtii hydA1-1hydA2-1.In contrast to the GFP transformants without the chloroplast transit peptide which showed the green fluorescence in the cytoplasm,the fluorescence from the cpTP-GFP transformants was distributed in the chloroplast.This further conformed the ability of C.reinaherdth HydAl chloroplast transit peptide of leading the fused protein into the chloroplast.Combined with the result from the parallel project "the homologous chloroplastic expression of HydA1 in the mutant hydA1-1hydA2-1" which proved that the codon optimized chydA1 according to the chloroplast bias could be expressed actively,it could be concluded that the FeS cluster of hydrogenase HydA1 is assembled in the C.reinhardtii chloroplast.