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 I-hydrogenase 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.

FIG. 1.

Models of the hydrogenase and photosystem I complexes used in this study. (A) Membrane-bound hydrogenase (MBH_wt) of Ralstonia eutropha H16. (B) Wild-type photosystem I (PS I) from Synechocystis sp. PCC 6803. (C) MBH_stop protein lacking the C-terminal anchor domain of HoxK. (D) MBH_PsaE and PS I_ΔPsaE.

FIG. 2.

Subcellular localization of HoxK and HoxG in R. eutropha H16 and mutant strains. Protein samples (cytoplasm, 20 μg [A]; membrane, 15 μg [B]; and periplasm, 20 μg [C]) were separated by SDS-PAGE. Western blot analysis was done using antibodies against HoxK, HoxG, and Strep-Tactin AP conjugate. HF210 (hydrogenase-free), HF689 (ΔHoxG), HF632 (MBH_wt), and HF768 (HoxK-PsaE-Strep-tag) were used.

FIG. 3.

Purification of MBH proteins from the membrane (ME) or from the soluble extract (SE). (A) In each lane, 5 μg protein was separated by SDS-PAGE and subsequently stained with Coomassie blue. (B) Specific proteins were identified immunologically. Lanes 1 show purification of MBH_wt (HF632) from the ME by using Ni-NTA affinity chromatography. Lanes 2 and 3 show purification of MBH_stop and MBH_PsaE* (HF653 and HF768, respectively) (see the text) via Strep-Tactin affinity chromatography from SE. Lanes 5 show purification of MBH_PsaE via Strep-Tactin affinity chromatography after removal of the HoxK-PsaE-HoxO/G/Q-His₆ complex (lanes 4) by immobilized-metal affinity chromatography (HF769). ▿, unknown ∼35-kDa protein.

FIG. 4.

Hydrophobic interaction chromatography (HIC) elution profile of PS I_ΔPsaE. The Ni-NTA eluate was complemented with a 3 M (NH₄)₂SO₄ solution at a volumetric ratio of 1:2. The resulting protein solution was loaded onto a HIC column (POROS 50 OH) which had been equilibrated with 20 mM HEPES, pH 7.5, 10 mM MgCl₂, 10 mM CaCl₂, 0.5 M mannitol, and 0.03% beta-DM. PS I derivatives were eluted by applying a linear gradient of 1.5 to 0 M (NH₄)₂SO₄. t, time; A, absorbance; a.u., arbitrary units.

FIG. 5.

Elution profiles of purified PS I_ΔPsaE trimers and monomers after size exclusion chromatography (see the text for details).

FIG. 6.

Purification of Strep-tagged PsaE protein via affinity chromatography. Cells were grown in FGN minimal medium, and after 24 h, gene expression was induced by the addition of 2 mM acetoine. The cells were grown for an additional 24 h and harvested, and soluble extract was prepared. The soluble extract was loaded onto a Strep-Tactin Superflow column. Proteins were separated by SDS-PAGE (Coomassie staining shown in panel A) and PsaE was detected with Strep-Tactin AP conjugate (B). Abbreviations: SE, soluble extract (20 μg); FT, flowthrough (20 μg); EL, eluate (5 μg).

FIG. 7.

In vitro separation of PS I_ΔPsaE-MBH_PsaE supercomplexes and trimeric PS I complexes by blue native PAGE. Ten picomoles of PS I_wt/PS I_ΔPsaE trimer, 40 pmol MBH_wt/MBH_stop/MBH_PsaE, and 400 pmol free PsaE were mixed, incubated for 30 min at 20°C, and subsequently loaded on a blue native gel.