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.
All Science Journal Classification (ASJC) codes
- Plant Science
- Cell Biology