Designing a modified clostridial 2[4Fe-4S] ferredoxin as a redox coupler to directly link photosystem I with a Pt nanoparticle.
Karim A WaltersJohn H GolbeckPublished in: Photosynthesis research (2019)
A methodology previously developed in our laboratory utilized an aliphatic hydrocarbon terminated by thiol groups to tether two redox proteins, i.e., the [4Fe-4S] cluster FB of photosystem I (PS I) and the distal [4Fe-4S] cluster of a [FeFe]-hydrogenase, to create a biohybrid dihydrogen-generating complex. These studies guided the design of a modified 2[4Fe-4S] cluster ferredoxin from Clostridium pasteurianum (CpFd) containing two externally facing cysteine residues in close proximity to each [4Fe-4S] cluster that replaces the aliphatic hydrocarbon dithiol tether. The advantage of using a protein is the potential to create a coupled dihydrogen-generating system in vivo. The wild-type CpFdWT and variants CpFdS11C/D40C, CpFdP20C/P49C, CpFdD7S/D36S, CpFdS11C/D40C/D7S/D36S and CpFdP20C/P49C/D7S/D36S were expressed in Escherichia coli and found to contain ~ 8 Fe and ~ 8 S atoms. The absorption spectra of the wild-type and CpFd variants displayed a peak centered at ~ 390 nm characteristic of a S → Fe charge transfer band that diminishes upon reduction with Na-dithionite. Low-temperature X-band EPR studies of the Na-dithionite-reduced wild-type and CpFd variants showed a complex spectrum indicative of two magnetically coupled [4Fe-4S]1+ clusters. EPR-monitored redox titrations of CpFdWT, CpFdD7S/D36S, CpFdS11C/D40C, CpFdP20C/P49C, CpFdS11C/D40C/D7S/D36S and CpFdP20C/P49C/D7S/D36S revealed redox potentials of - 412 ± 8 mV, - 395 ± 4 mV, - 408 ± 7 mV, - 426 ± 11 mV, - 384 ± 4 mV and - 423 ± 4 mV, respectively. The in vitro PS I-CpFdS11C/D40C/D7S/D36S-Pt nanoparticle complex was the highest performer, generating dihydrogen at a rate of 3.25 μmol H2 mg Chl-1 h-1 or 278.8 mol H2 mol PS I-1 h-1 under continuous illumination.