Presence of a [3Fe-4S] cluster in a PsaC variant as a functional component of the photosystem I electron transfer chain in Synechococcus sp. PCC 7002.

A site-directed C14G mutation was introduced into the stromal PsaC subunit of Synechococcus sp. strain PCC 7002 in vivo in order to introduce an exchangeable coordination site into the terminal FB [4Fe-4S] cluster of Photosystem I (PSI). Using an engineered PSI-less strain (psaAB deletion), psaC was deleted and replaced with recombinant versions controlled by a strong promoter, and the psaAB deletion was complemented. Modified PSI accumulated at lower levels in this strain and supported slower photoautotrophic growth than wild type. As-isolated PSI complexes containing PsaCC14G showed resonances with g values of 2.038 and 2.007 characteristic of a [3Fe-4S]1+ cluster. When the PSI complexes were illuminated at 15 K, these resonances partially disappeared and two new sets of resonances appeared. The majority set had g values of 2.05, 1.95, and 1.85, characteristic of FA-, and the minority set had g values of 2.11, 1.90, and 1.88 from FB' in the modified site. The S = 1/2 spin state of the latter implied the presence of a thiolate as the terminal ligand. The [3Fe-4S] clusters could be partially reconstituted with iron, producing a larger population of [4Fe-4S] clusters. Rates of flavodoxin reduction were identical in PSI complexes isolated from wild type and the PsaCC14G variant strain; this implied equivalent capacity for forward electron transfer in PSI complexes that contained [3Fe-4S] and [4Fe-4S] clusters. The development of this cyanobacterial strain is a first step toward translation of in vitro PSI-based biosolar molecular wire systems in vivo and provides new insights into the formation of Fe/S clusters.

Quantum yield measurements of light-induced H2 generation in a photosystem I-[FeFe]-H2ase nanoconstruct.

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.

Solar hydrogen-producing bionanodevice outperforms natural photosynthesis.

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.