Requirements for Construction of a Functional Hybrid Complex of Photosystem I and [NiFe]-Hydrogenase

Abstract

The development of cellular systems in which the enzyme hydrogenase is efficiently coupled to the oxygenic photosynthesis apparatus represents an attractive avenue to produce H 2 sustainably from light and water. Here we describe the molecular design of the individual components required for the direct coupling of the O 2 -tolerant membrane-bound hydrogenase (MBH) from Ralstonia eutropha H16 to the acceptor site of photosystem I (PS I) from Synechocystis sp. PCC 6803. By genetic engineering, the peripheral subunit PsaE of PS I was fused to the MBH, and the resulting hybrid protein was purified from R. eutropha to apparent homogeneity via two independent affinity chromatographical steps. The catalytically active MBH-PsaE (MBH PsaE ) hybrid protein could be isolated only from the cytoplasmic fraction. This was surprising, since the MBH is a substrate of the twin-arginine translocation system and was expected to reside in the periplasm. We conclude that the attachment of the additional PsaE domain to the small, electron-transferring subunit of the MBH completely abolished the export competence of the protein. Activity measurements revealed that the H 2 production capacity of the purified MBH PsaE fusion protein was very similar to that of wild-type MBH. In order to analyze the specific interaction of MBH PsaE with PS I, His-tagged PS I lacking the PsaE subunit was purified via Ni-nitrilotriacetic acid affinity and subsequent hydrophobic interaction chromatography. Formation of PS Ihydrogenase supercomplexes was demonstrated by blue native gel electrophoresis. The results indicate a vital prerequisite for the quantitative analysis of the MBH PsaE -PS I complex formation and its light-driven H 2 production capacity by means of spectroelectrochemistry.

The Hydrogenase Subcomplex of the NAD+‐Reducing [NiFe] Hydrogenase from Ralstonia eutropha – Insights into Catalysis and Redox Interconversions

“…These differences are likely to be related to the missing FMN and/or the lack of the diaphorase moiety, suggesting a major influence of the electron pathway on the redox behavior of the [NiFe] [45] Hydrogen production was quantified at 30°C using a modified Clark electrode and reduced MV as the electron donor. [46] The assay chamber was filled with 1.3 mL of N 2 -saturated Tris-HCl buffer (50 mm pH 8.0) containing 7.7 mm dithionite and 5 mm MV. For hydrogen oxidation activity, the reduction of MV (5 mm) in 2 mL of H 2 -saturated Tris-HCl buffer (50 mm, pH 8.0) was monitored at 30°C and 605 nm (the molar extinction coefficient of MV is 1.3 ϫ 10 4 m -1 cm -1 [47] ).…”

Development of a Transferable Bimolecular Fluorescence Complementation System for the Investigation of Interactions between Poly(3-Hydroxybutyrate) Granule-Associated Proteins in Gram-Negative Bacteria

“…The rrnB-P BAD -eyfp-c1 and rrnB-P BAD -yc-c1 cassettes were amplified from pBBR-EYFP-C1 and pBBR-YC-C1 using the primers rrnB_fwd_NsiI and EYFP-C1_r_SpeI, cut with SphI and SpeI (the rrnB_fwd_NsiI primer also contained a SphI site), and subcloned into a derivative of pCM62 (see below) that had been cut with the same enzymes. The plasmid pLO11 (a derivative of pCM62 [26]) was cut with AflIII and EcoRI to obtain the pCM62 backbone and ligated with annealed oligonucleotides AdapA and AdapB, which contain internal SphI and SpeI sites for subcloning of fragments from pBBR1MCS-2-based plasmids in pCM62. The resulting plasmids were designated pCM-EYFP-C1 and pCM-YC-C1.…”

Oxygen-tolerant hydrogenases in hydrogen-based technologies

“…Upon illumination, the in vitro reconstructed PS I-MBH fusion protein generates H 2 at unexpectedly low rates [0.58 mmol of H 2 (mg chlorophyll) À1 h À1 ] [44 ]. However, a reconstructed, highly purified PS I-MBH hybrid complex [46], assembled on a gold electrode (Figure 2b), increased H 2 production rates substantially [3000 mmol of H 2 (mg chlorophyll) À1 h À1 ] [43 ]. This observation indicates that the motion of the individual protein modules in solution represents a severe rate-limiting step that is obviously circumvented by immobilizing the protein complexes on solid surfaces mimicking the in vivo situation of the membrane-associated enzymes.…”