ABSTRACT
The quantum yield for light-induced H2 generation was measured for a previously optimized bio-hybrid cytochrome c 6-crosslinked PSI(C13G)-1,8-octanedithiol-[FeFe]-H2ase(C97G) (PSI-H2ase) nanoconstruct. The theoretical quantum yield for the PSI-H2ase nanoconstruct is 0.50 molecules of H2 per photon absorbed, which equates to a requirement of two photons per H2 generated. Illumination of the PSI-H2ase nanoconstruct with visible light between 400 and 700 nm resulted in an average quantum yield of 0.10-0.15 molecules of H2 per photon absorbed, which equates to a requirement of 6.7-10 photons per H2 generated. A possible reason for the difference between the theoretical and experimental quantum yield is the occurrence of non-productive PSI(C13G)-1,8-octanedithiol-PSIC13G (PSI-PSI) conjugates, which would absorb light without generating H2. Assuming the thiol-Fe coupling is equally efficient at producing PSI-PSI conjugates as well as in producing PSI-H2ase nanoconstructs, the theoretical quantum yield would decrease to 0.167 molecules of H2 per photon absorbed, which equates to 6 photons per H2 generated. This value is close to the range of measured values in the current study. A strategy that purifies the PSI-H2ase nanoconstructs from the unproductive PSI-PSI conjugates or that incorporates different chemistries on the PSI and [FeFe]-H2ase enzyme sites could potentially allow the PSI-H2ase nanoconstruct to approach the expected theoretical quantum yield for light-induced H2 generation.
Subject(s)
Hydrogen/metabolism , Nanostructures/chemistry , Photosystem I Protein Complex/metabolism , Biofuels , Cross-Linking Reagents/chemistry , Cytochromes c6/chemistry , Cytochromes c6/metabolism , Hydrogenase/chemistry , Hydrogenase/metabolism , Iron/chemistry , Iron/metabolism , Light , Photosystem I Protein Complex/chemistry , Quantum Theory , Sulfhydryl Compounds/chemistryABSTRACT
The active center (H-cluster) of [FeFe]-hydrogenases is embedded into a hydrophobic pocket within the protein. We analyzed several amino acids, located in the vicinity of this niche, by site-directed mutagenesis of the [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA1). These amino acids are highly conserved and predicted to be involved in H-cluster coordination. Characterization of two hydrogenase variants confirmed this hypothesis. The exchange of residues CrHydA1Met(415) and CrHydA1Lys(228) resulted in inactive proteins, which, according to EPR and FTIR analyses, contain no intact H-cluster. However, [FeFe]-hydrogenases in which CpIMet(353) (CrHydA1Met(223)) and CpICys(299) (CrHydA1Cys(169)) were exchanged to leucine and serine, respectively, showed a structurally intact H-cluster with catalytic activity either absent (CpIC299S) or strongly diminished (CpIM353L). In the case of CrHydA1C169S, the H-cluster was trapped in an inactive state exhibiting g values and vibrational frequencies that resembled the H(trans) state of DdH from Desulfovibrio desulfuricans. This cysteine residue, interacting with the bridge head nitrogen of the di(methyl)amine ligand, seems therefore to represent an essential contribution of the immediate protein environment to the reaction mechanism. Exchanging methionine CpIM(353) (CrHydA1M(223)) to leucine led to a strong decrease in turnover without affecting the K(m) value of the electron donor. We suggest that this methionine constitutes a "fine-tuning" element of hydrogenase activity.
Subject(s)
Bacterial Proteins/chemistry , Chlamydomonas reinhardtii/enzymology , Clostridium/enzymology , Desulfovibrio desulfuricans/enzymology , Hydrogenase/chemistry , Plant Proteins/chemistry , Bacterial Proteins/genetics , Catalysis , Chlamydomonas reinhardtii/genetics , Clostridium/genetics , Desulfovibrio desulfuricans/genetics , Electron Spin Resonance Spectroscopy , Hydrogenase/genetics , Kinetics , Mutagenesis, Site-Directed , Plant Proteins/genetics , Spectroscopy, Fourier Transform InfraredABSTRACT
Although a number of solar biohydrogen systems employing photosystem I (PSI) have been developed, few attain the electron transfer throughput of oxygenic photosynthesis. We have optimized a biological/organic nanoconstruct that directly tethers F(B), the terminal [4Fe-4S] cluster of PSI from Synechococcus sp. PCC 7002, to the distal [4Fe-4S] cluster of the [FeFe]-hydrogenase (H(2)ase) from Clostridium acetobutylicum. On illumination, the PSI-[FeFe]-H(2)ase nanoconstruct evolves H(2) at a rate of 2,200 ± 460 µmol mg chlorophyll(-1) h(-1), which is equivalent to 105 ± 22 e(-)PSI(-1) s(-1). Cyanobacteria evolve O(2) at a rate of approximately 400 µmol mg chlorophyll(-1) h(-1), which is equivalent to 47 e(-)PSI(-1) s(-1), given a PSI to photosystem II ratio of 1.8. The greater than twofold electron throughput by this hybrid biological/organic nanoconstruct over in vivo oxygenic photosynthesis validates the concept of tethering proteins through their redox cofactors to overcome diffusion-based rate limitations on electron transfer.
Subject(s)
Clostridium acetobutylicum/metabolism , Hydrogen/metabolism , Nanostructures , Nanotechnology/methods , Photosynthesis , Solar Energy , Synechococcus/metabolism , Electron Transport , Iron/metabolism , Kinetics , Photosystem I Protein Complex/metabolism , Spectrum AnalysisABSTRACT
The cell wall of Nocardia farcinica contains a cation-selective cell wall channel, which may be responsible for the limited permeability of the cell wall of N. farcinica for negatively charged antibiotics. Based on partial sequencing of the protein responsible for channel formation derived from N. farcinica ATTC 3318 we were able to identify the corresponding genes (nfa15890 and nfa15900) within the known genome of N. farcinica IFM 10152. The corresponding genes of N. farcinica ATTC 3318 were separately expressed in the Escherichia coli BL21DE3Omp8 strain and the N-terminal His10-tagged proteins were purified to homogeneity using immobilized metal affinity chromatography. The pure proteins were designated NfpANHis and NfpBNHis, for N. farcinica porin A and N. farcinica porin B. The two proteins were checked separately for channel formation in lipid bilayers. Our results clearly indicate that the proteins NfpANHis and NfpBNHis expressed in E. coli could only together form a channel in lipid bilayer membranes. This means that the cell wall channel of N. farcinica is formed by a heterooligomer. NfpA and NfpB form together a channel that may structurally be related to MspA of Mycobacterium smegmatis based on amino acid comparison and renaturation procedure.
Subject(s)
Bacterial Proteins/chemistry , Nocardia/metabolism , Porins/chemistry , Protein Multimerization , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Blotting, Western , Cell Wall/metabolism , Cloning, Molecular , Hydrogen-Ion Concentration , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Models, Molecular , Molecular Sequence Data , Nocardia/genetics , Porins/genetics , Porins/metabolism , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Analysis, Protein , Sequence Homology, Amino AcidABSTRACT
A molecular wire is used to connect two proteins through their physiologically relevant redox cofactors to facilitate direct electron transfer. Photosystem I (PS I) and an [FeFe]-hydrogenase (H(2)ase) serve as the test bed for this new technology. By tethering a photosensitizer with a hydrogen-evolving catalyst, attached by Fe-S coordination bonds between the F(B) iron-sulfur cluster of PS I and the distal iron-sulfur cluster of H(2)ase, we assayed electron transfer between the two components via light-induced hydrogen generation. These hydrogen-producing nanoconstructs self-assemble when the PS I variant, the H(2)ase variant, and the molecular wire are combined.
