RESUMEN
Active [FeFe] hydrogenases can be obtained by expressing the unmaturated enzyme in Escherichia coli followed by incubation with a synthetic precursor of the binuclear [2Fe] subcluster, namely: [NEt4]2[Fe2(adt)(CO)4(CN)2] (adt = [S-CH2-NH-CH2-S](2-)). The binuclear subsite Fe2(adt)(CO)3(CN)2 is attached through a bridging cysteine side chain to a [4Fe-4S] subcluster already present in the unmaturated enzyme thus yielding the intact native "H-cluster". We present FTIR electrochemical studies of the [FeFe] hydrogenase from Chlamydomonas reinhardtii, CrHydA1, maturated with the precursor of the native cofactor [Fe2(adt)(CO)4(CN)2](2-) as well as a non-natural variant [Fe2(pdt)(CO)4(CN)2](2-) in which the bridging amine functionality is replaced by CH2. The obtained active enzyme CrHydA1(adt) shows the same redox states in the respective potential range as observed for the native system (E(ox/red) = -400 mV, E(red/sred) = -470 mV). For the Hox â Hred transition the reducing equivalent is stored on the binuclear part, ([4Fe-4S](2+)Fe(II)Fe(I) â [4Fe-4S](2+)Fe(I)Fe(I)), while the Hred â Hsred transition is characterized by a reduction of the [4Fe-4S] part of the H-cluster ([4Fe-4S](2+)Fe(I)Fe(I) â [4Fe-4S](+)Fe(I)Fe(I)). A similar transition is reported here for the CO inhibited state of the H-cluster: ([4Fe-4S](2+)Fe(I)Fe(II)CO â [4Fe-4S](+)Fe(I)Fe(II)CO). An FTIR electrochemical study of the inactive variant with the pdt ligand, CrHydA1(pdt), identified two redox states H(pdt)-ox and H(pdt)-"red". Both EPR and FTIR spectra of H(pdt)-ox are virtually identical to those of the H(adt)-ox and the native Hox state. The H(pdt)-"red" state is also characterized by a reduced [4Fe-4S] subcluster. In contrast to CrHydA1(adt), the H(pdt)-ox state of CrHydA1(pdt) is stable up to rather high potentials (+200 mV). This study demonstrates the distinct redox coupling between the two parts of the H-cluster and confirms that the [4Fe-4S]H subsite is also redox active and as such an integral part of the H-cluster taking part in the catalytic cycle.
Asunto(s)
Hidrógeno/química , Hidrogenasas/química , Hierro/química , Oxidación-Reducción , Aminas/química , Catálisis , Dominio Catalítico , Chlamydomonas reinhardtii/enzimología , Análisis por Conglomerados , Cisteína/química , Electroquímica , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/enzimología , Ligandos , Protones , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
In anaerobiosis, the green alga Chlamydomonas reinhardtii evolves molecular hydrogen (H(2)) as one of several fermentation products. H(2) is generated mostly by the [Fe-Fe]-hydrogenase HYDA1, which uses plant type ferredoxin PETF/FDX1 (PETF) as an electron donor. Dark fermentation of the alga is mainly of the mixed acid type, because formate, ethanol, and acetate are generated by a pyruvate:formate lyase pathway similar to Escherichia coli. However, C. reinhardtii also possesses the pyruvate:ferredoxin oxidoreductase PFR1, which, like pyruvate:formate lyase and HYDA1, is localized in the chloroplast. PFR1 has long been suggested to be responsible for the low but significant H(2) accumulation in the dark because the catalytic mechanism of pyruvate:ferredoxin oxidoreductase involves the reduction of ferredoxin. With the aim of proving the biochemical feasibility of the postulated reaction, we have heterologously expressed the PFR1 gene in E. coli. Purified recombinant PFR1 is able to transfer electrons from pyruvate to HYDA1, using the ferredoxins PETF and FDX2 as electron carriers. The high reactivity of PFR1 toward oxaloacetate indicates that in vivo, fermentation might also be coupled to an anaerobically active glyoxylate cycle. Our results suggest that C. reinhardtii employs a clostridial type H(2) production pathway in the dark, especially because C. reinhardtii PFR1 was also able to allow H(2) evolution in reaction mixtures containing Clostridium acetobutylicum 2[4Fe-4S]-ferredoxin and [Fe-Fe]-hydrogenase HYDA.
Asunto(s)
Chlamydomonas reinhardtii/enzimología , Proteínas de Cloroplastos/metabolismo , Cloroplastos/enzimología , Hidrógeno/metabolismo , Piruvato-Sintasa/metabolismo , Chlamydomonas reinhardtii/genética , Proteínas de Cloroplastos/genética , Cloroplastos/genética , Ferredoxinas/genética , Ferredoxinas/metabolismo , Hidrogenasas/genética , Hidrogenasas/metabolismo , Piruvato-Sintasa/genéticaRESUMEN
In the present investigation, a detailed biochemical analysis of the high H2 producer D1 protein mutant strain L159I-N230Y of Chlamydomonas reinhardtii, carrying a double amino acid substitution, was made. The leucine residue L159 was replaced by isoleucine, and the N230 asparagine was replaced by tyrosine. The performance of this strain was compared to that of the cc124 strain. The mutant showed a sustained capacity to donate electrons by means of direct biophotolysis for H2 production, as demonstrated by the higher efficiency of utilization of the hydrogenase enzyme when carried out under anaerobic conditions. The latter property was maintained also under sulfur deprivation. Furthermore, when compared to the cc124, the mutant showed a higher amount of D1 protein content, a higher carbohydrate storage capacity and a sustained PSII direct contribution to the H2 production during sulfur deprivation. The addition of DCMU to the cells showed that as much as 7.0 mL H2 liter of culture h⻹ were produced by means of direct biophotolysis. The maximum apparent light-to-hydrogen conversion efficiency expressed on PAR (photosynthetically active radiation) reached 3.22%, while PSII efficiency to perform direct biophotolysis was calculated to be 2.03%. These values are significantly higher than what has been reported in the literature.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Hidrógeno/metabolismo , Mutación/genética , Complejo de Proteína del Fotosistema II/genética , Western Blotting , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Chlamydomonas reinhardtii/efectos de los fármacos , Chlamydomonas reinhardtii/enzimología , Diurona/farmacología , Hidrogenasas/metabolismo , Procesos Fotoquímicos/efectos de los fármacos , Fotosíntesis/efectos de los fármacos , Complejo de Proteína del Fotosistema II/inmunología , Azufre/deficienciaRESUMEN
Hydrogen photoproduction by eukaryotic microalgae results from a connection between the photosynthetic electron transport chain and a plastidial hydrogenase. Algal H2 production is a transitory phenomenon under most natural conditions, often viewed as a safety valve protecting the photosynthetic electron transport chain from overreduction. From the colony screening of an insertion mutant library of the unicellular green alga Chlamydomonas reinhardtii based on the analysis of dark-light chlorophyll fluorescence transients, we isolated a mutant impaired in cyclic electron flow around photosystem I (CEF) due to a defect in the Proton Gradient Regulation Like1 (PGRL1) protein. Under aerobiosis, nonphotochemical quenching of fluorescence (NPQ) is strongly decreased in pgrl1. Under anaerobiosis, H2 photoproduction is strongly enhanced in the pgrl1 mutant, both during short-term and long-term measurements (in conditions of sulfur deprivation). Based on the light dependence of NPQ and hydrogen production, as well as on the enhanced hydrogen production observed in the wild-type strain in the presence of the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, we conclude that the proton gradient generated by CEF provokes a strong inhibition of electron supply to the hydrogenase in the wild-type strain, which is released in the pgrl1 mutant. Regulation of the trans-thylakoidal proton gradient by monitoring pgrl1 expression opens new perspectives toward reprogramming the cellular metabolism of microalgae for enhanced H2 production.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Electrones , Hidrógeno/metabolismo , Fotosíntesis/fisiología , Proteínas de Plantas/metabolismo , Protones , Aerobiosis , Anaerobiosis , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Chlamydomonas reinhardtii/citología , Chlamydomonas reinhardtii/genética , Transporte de Electrón/efectos de los fármacos , Transporte de Electrón/fisiología , Prueba de Complementación Genética , Hidrogenasas/metabolismo , Luz , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Fotosíntesis/efectos de los fármacos , Complejo de Proteína del Fotosistema I/efectos de los fármacos , Complejo de Proteína del Fotosistema I/genética , Complejo de Proteína del Fotosistema I/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Ionóforos de Protónes/farmacología , Azufre/metabolismoRESUMEN
The green alga Chlamydomonas reinhardtii has a complex anaerobic metabolism characterized by a plastidic hydrogenase (HYD1) coupled to photosynthesis and a bacterial-type fermentation system in which pyruvate formate lyase (PFL1) is the central fermentative enzyme. To identify mutant strains with altered hydrogen metabolism, a C. reinhardtii nuclear transformant library was screened. Mutant strain 48F5 showed lower light-dependent hydrogen (H2) evolution rates and reduced in vitro hydrogenase activity, and fermentative H2 production in the dark was enhanced. The transformant has a single integration of the paromomycin resistance cassette within the PFL1 gene, and is unable to synthesize PFL1 protein. 48F5 secretes no formate, but produces more ethanol, D-lactate and CO2 than the wild type. Moreover, HYD1 transcript and HYD1 protein levels were lower in the pfl1 mutant strain. Complementation of strain 48F5 with an intact copy of the PFL1 gene restored formate excretion and hydrogenase activity to the wild type level. This analysis shows that the PFL1 pathway has a significant impact on hydrogen metabolism in C. reinhardtii.