Rational redesign of the ferredoxin-NADP+-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H2-production

Abstract

Utilization of electrons from the photosynthetic water splitting reaction for the generation of biofuels, commodities as well as application in biotransformations requires a partial rerouting of the photosynthetic electron transport chain. Due to its rather negative redox potential and its bifurcational function, ferredoxin at the acceptor side of Photosystem 1 is one of the focal points for such an engineering. With hydrogen production as model system, we show here the impact and potential of redox partner design involving ferredoxin (Fd), ferredoxin-oxido-reductase (FNR) and [FeFe]‑hydrogenase HydA1 on electron transport in a future cyanobacterial design cell of Synechocystis PCC 6803. X-ray-structure-based rational design and the allocation of specific interaction residues by NMR-analysis led to the construction of Fd- and FNR-mutants, which in appropriate combination enabled an about 18-fold enhanced electron flow from Fd to HydA1 (in competition with equimolar amounts of FNR) in in vitro assays. The negative impact of these mutations on the Fd-FNR electron transport which indirectly facilitates H production (with a contribution of ≤42% by FNR variants and ≤23% by Fd-variants) and the direct positive impact on the Fd-HydA1 electron transport (≤23% by Fd-mutants) provide an excellent basis for the construction of a hydrogen-producing design cell and the study of photosynthetic efficiency-optimization with cyanobacteria.

Solvent dynamics play a decisive role in the complex formation of biologically relevant redox proteins

“…S1, ESI †). 16,17,44 Neutralization of these surface charges upon complexation results in a change in the surface electrostatic potential such that the solute is overall neutral. Our results demonstrate that the THz responses of FNR, PetF, and the FNR:PetF complex are clearly distinguishable.…”

“…Protein expression and purification FNR, PetF, and FDX3 proteins were expressed and purified according to previously established protocols (see ESI † for details). 16 All protein samples were stored at À80 1C until further use. Specific concentrations were prepared from dilution of the stock solution.…”

“…S1 in the ESI †). 13,[15][16][17] This transient complex is further stabilized by several amino acids from both proteins that form a hydrophobic environment near the two redox centers which were reported to be in close proximity to each other (B6 Å) to ensure fast electron transfer (Fig. S2, ESI †).…”

Biocatalysis and Strategies for Enzyme Improvement

“…RR strategies for enzyme improvement genetically modify the existing biocatalyst based on known structural criteria. To begin with, different pieces of data, like protein structure obtained by X-ray diffraction techniques, molecular models based on computational algorithms, and biochemical specifics, like locations of interacting ligand residues obtained by NMR-analysis, must be evaluated to propose rational genetic alterations [94]. The method of choice for introducing specific alterations is site-directed mutagenesis (SDM) [2].…”

“…A more efficient one-step version of QuikChange method suitable for single or multiple-site insertions and deletions was described in [96]. Successful applications of SDM include the induction of acid-resistance and 16.7-fold higher catalytic activity in a-amylase used for industrial sugar and detergent production [97], thermostability enhancement in sucrose isomerase producing a non-cariogenic, nutritional sugar isomaltulose that slows down the rate of insulin release in diabetes management [98], and even attempts to design high efficiency H 2 -producing cyanobacterial cells to make biofuels [94]. At the same time, numerous attempts of RR fail due to insufficient knowledge of mechanisms responsible for the specific structure-function relationships.…”