Optimized over-expression of [FeFe] hydrogenases with high specific activity in Clostridium acetobutylicum

Abendroth, Gregory von; Stripp, Sven T.; Silakov, Alexey; Croux, Christian; Soucaille, Philippe; Girbal, Laurence; Happe, Thomas

doi:10.1016/j.ijhydene.2008.07.122

Cited by 78 publications

(70 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The [FeFe]-hydrogenases CaHydA and CrHydA1 were isolated as described previously (36) as were the [NiFe]-hydrogenases EcHyd1 and EcHyd2 (31). Experiments were carried out in a glovebox under N 2 (O 2 < 2 ppm).…”

Section: Methodsmentioning

confidence: 99%

Electrocatalytic mechanism of reversible hydrogen cycling by enzymes and distinctions between the major classes of hydrogenases

Hexter

Grey

Happe

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

160

223

View full text Add to dashboard Cite

The authors note that on page 11517, right column, fourth full paragraph, lines 4-5, "k min = k 0 and k max = k 0 exp(−βd 0 )" should instead appear as "k max = k 0 and k min = k 0 exp(−βd 0 )." Also, on page 11518, left column, fourth full paragraph, line 8, "e 2 = k 2a /k 2c " should instead appear as "e 2 = k 2c /k 2a ."Figs. 4, 5, and 6 and the legend for Figure 5 appeared incorrectly. The corrected figures and their legends appear below.

show abstract

Section: Methodsmentioning

confidence: 99%

Electrocatalytic mechanism of reversible hydrogen cycling by enzymes and distinctions between the major classes of hydrogenases

Hexter

Grey

Happe

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

160

223

View full text Add to dashboard Cite

show abstract

“…Recombinant C. reinhardtii [FeFe] hydrogenase CrHydA1 was synthesized and isolated anaerobically from C. acetobutylicum as previously described (38). To prepare samples for XAS, isolated protein was concentrated to 1 mM (48 g/L) by using Vivaspin 6 and Vivaspin 500 columns (Sartorius Stedim Biotech) and stored in 0.1 mM Tris/HCl pH 8.0, 2 mM sodium dithionite (NaDT), and 10% glycerol.…”

Section: Methodsmentioning

confidence: 99%

How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms

Stripp

Goldet

Brandmayr

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

317

323

View full text Add to dashboard Cite

Green algae such as Chlamydomonas reinhardtii synthesize an [FeFe] hydrogenase that is highly active in hydrogen evolution. However, the extreme sensitivity of [FeFe] hydrogenases to oxygen presents a major challenge for exploiting these organisms to achieve sustainable photosynthetic hydrogen production. In this study, the mechanism of oxygen inactivation of the [FeFe] hydrogenase CrHydA1 from C. reinhardtii has been investigated. X-ray absorption spectroscopy shows that reaction with oxygen results in destruction of the [4Fe-4S] domain of the active site H-cluster while leaving the di-iron domain (2FeH) essentially intact. By protein film electrochemistry we were able to determine the order of events leading up to this destruction. Carbon monoxide, a competitive inhibitor of CrHydA1 which binds to an Fe atom of the 2FeH domain and is otherwise not known to attack FeS clusters in proteins, reacts nearly two orders of magnitude faster than oxygen and protects the enzyme against oxygen damage. These results therefore show that destruction of the [4Fe-4S] cluster is initiated by binding and reduction of oxygen at the di-iron domain-a key step that is blocked by carbon monoxide. The relatively slow attack by oxygen compared to carbon monoxide suggests that a very high level of discrimination can be achieved by subtle factors such as electronic effects (specific orbital overlap requirements) and steric constraints at the active site.EXAFS ͉ H-cluster ͉ protein film electrochemistry ͉ biological hydrogen production ͉ green algae

show abstract

“…However, in contrast to the well studied interaction of PetF with other redox partners like ferredoxin-NADPH oxidoreductase (14,15), the mechanism of the electron transfer process between PetF and the algal hydrogenase is still an open question (9,10,16). Recently, we reported the establishment of an efficient system for the heterologous synthesis of [FeFe] hydrogenases, including HydA1 of C. reinhardtii (17). Using this system, we generated several variants of HydA1 and PetF that were specifically designed on the basis of predicted electrostatic surface distribution and preceding in silico docking analyses.…”

mentioning

confidence: 99%

“…The experimental data demonstrate that especially Lys 396 of HydA1, which is particularly conserved among green algal hydrogenases, is crucial for a successful binding and electron transfer between PetF and HydA1. demonstrated to allow efficient heterologous expression in the Clostridium acetobutylicum host strain ATCC 824 (17). SDM variants of cloned cDNA sequences from hydA1 and petF1 were created in a two-step procedure using mismatch primers according to the fusion PCR technique (18).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Characterization of the Key Step for Light-driven Hydrogen Evolution in Green Algae

Winkler

Kuhlgert

Hippler

et al. 2009

Journal of Biological Chemistry

115

View full text Add to dashboard Cite

Under anaerobic conditions, several species of green algae perform a light-dependent hydrogen production catalyzed by a special group of [FeFe] hydrogenases termed HydA. Although highly interesting for biotechnological applications, the direct connection between photosynthetic electron transport and hydrogenase activity is still a matter of speculation. By establishing an in vitro reconstitution system, we demonstrate that the photosynthetic ferredoxin (PetF) is essential for efficient electron transfer between photosystem I and HydA1. To investigate the electrostatic interaction process and electron transfer between PetF and HydA1, we performed site-directed mutagenesis. Kinetic analyses with several site-directed mutagenesis variants of HydA1 and PetF enabled us to localize the respective contact sites. These experiments in combination with in silico docking analyses indicate that electrostatic interactions between the conserved HydA1 residue Lys 396 and the C terminus of PetF as well as between the PetF residue Glu 122 and the N-terminal amino group of HydA1 play a major role in complex formation and electron transfer. Mapping of relevant HydA1 and PetF residues constitutes an important basis for manipulating the physiological photosynthetic electron flow in favor of light-driven H 2 production.Among all photosynthetic organisms, only green algae can couple light-driven electron transport originating from water splitting with hydrogen production (1). Hydrogen evolution in the unicellular green alga Chlamydomonas reinhardtii is naturally induced upon nutrient deprivation (2). Especially in the absence of sulfur, the photosynthetic oxygen evolution rate drops below the respiratory rate leading to intracellular anaerobiosis. Under anaerobic conditions, the oxygen-sensitive (2, 3) [FeFe] hydrogenase HydA is synthesized and catalyzes light-dependent H 2 production, thereby dissipating excess redox equivalents under conditions in which the Calvin cycle is down-regulated (4).The extraordinarily small monomeric [FeFe] hydrogenases of green algae only consist of the catalytic core unit containing the active site (H-cluster), whereas other [FeFe] hydrogenases possess an additional N-terminal F-domain harboring one to four accessory iron-sulfur clusters (5, 6). Because Chlorophytatype [FeFe] hydrogenases lack any accessory clusters (7, 8), a direct electron transfer between the native electron donor and the H-cluster has been assumed (9, 10). In C. reinhardtii, HydA1 has been shown to be localized in the chloroplast stroma (11), and first kinetic examinations with purified proteins demonstrated that the plastidic ferredoxin PetF can interact with HydA1. These results and the fact that H 2 production in C. reinhardtii is photosystem I (PSI) 2 -dependent (4) led to the hypothesis that PetF is the native electron donor of the plastidic hydrogenase (12). Derived from in silico analyses, two possible PetF-HydA2 electron transfer complex models were recently suggested (13). However, in contrast to the well studied interacti...

show abstract

Optimized over-expression of [FeFe] hydrogenases with high specific activity in Clostridium acetobutylicum

Cited by 78 publications

References 25 publications

Electrocatalytic mechanism of reversible hydrogen cycling by enzymes and distinctions between the major classes of hydrogenases

Electrocatalytic mechanism of reversible hydrogen cycling by enzymes and distinctions between the major classes of hydrogenases

How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms

Characterization of the Key Step for Light-driven Hydrogen Evolution in Green Algae

Contact Info

Product

Resources

About