Contents lists available at ScienceDirect BBA - Bioenergetics journal homepage: www.elsevier.com/locate/bbabio Rational redesign of the ferredoxin-NADP + -oxido-reductase/ferredoxin- interaction for photosynthesis-dependent H 2 -production Wiegand K. a,1 , Winkler M. b,1 , Rumpel S. c,1 , Kannchen D. a , Rexroth S. a , Hase T. d , Farès C. e , Happe T. b , Lubitz W. c , Rögner M. a, ⁎ a Plant Biochemistry, Faculty of Biology & Biotechnology, Ruhr University Bochum, 44780 Bochum, Germany b Photobiotechnology, Faculty of Biology & Biotechnology, Ruhr University Bochum, 44780 Bochum, Germany c Max-Planck-Institut für Chemische Energiekonversion, 45470 Mülheim, Germany d Institute for Protein Research, Osaka University, Suita 565-0871, Osaka, Japan e Max-Planck-Institut für Kohlenforschung, 45470 Mülheim, Germany ARTICLE INFO Keywords: Protein design Protein interaction FNR NMR Photosynthesis Synechocystis ABSTRACT Utilization of electrons from the photosynthetic water splitting reaction for the generation of biofuels, com- modities 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 ac- ceptor 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), fer- redoxin-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 in- teraction 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 2 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 effi- ciency-optimization with cyanobacteria. 1. Introduction Cyanobacteria harvest light energy with particular efficiency and transform it into chemical energy - a process which goes along with CO 2 -fixation and oxygen release. Due to the many steps involved in this process, the final efficiency of photosynthesis - based on the output of sugar or related compounds - is very low, routinely below 1% [1]. It is therefore tempting to modify the natural cyanobacterial metabolism to shorten the process of energy transformation as much as possible in order to increase the efficiency and to capture compounds suitable for long-term energy storage directly from photosynthesis. Hydrogen (H 2 ) fulfills these requirements and is one of the possible photosynthetic products of both, cyanobacteria [2] and green algae [3,4]. Besides its high energy content and unmatched environmental sustainability, H 2 is easily harvested due to its release from the cells into the medium. The S- deprivation metabolism of green algae like Chlamydomonas reinhardtii provides an impressive natural example for an enduring and productive photohydrogen evolution activity based on the [FeFe]‑hydrogenase HydA1; remarkably, this enzyme is directly coupled to the photo- synthetic electron transport via the plant-type ferredoxin PetF [5,6]. Several trials to implement and optimize an [FeFe]‑hydrogenase-based photohydrogen metabolism in cyanobacteria emulating the example of C. reinhardtii have been made [7,8]. Among them, the heterologous expression of a clostridial [FeFe]‑hydrogenase in Synechococcus elon- gatus sp. 7942 and its coupling to Photosystem 1 (PS1) via Fd of the host was most successful [9]. While photohydrogen evolution clearly ex- ceeded the H 2 -production activity of the native [NiFe]‑hydrogenase, it did not reach the level of anaerobic C. reinhardtii cultures. This is https://doi.org/10.1016/j.bbabio.2018.01.006 Received 5 September 2017; Received in revised form 18 January 2018; Accepted 22 January 2018 ⁎ Corresponding author. 1 Authors contributed equally. E-mail address: matthias.roegner@rub.de (M. Rögner). Abbreviations: C. reinhardtii, Chlamydomonas reinhardtii; Fd, PetF, ferredoxin; FNR, ferredoxin-NADP + -reductase; HydA1, [FeFe]‑hydrogenase of Chlamydomonas reinhardtii; S. 6803, Synechocystis PCC 6803; PS1, Photosystem 1 BBA - Bioenergetics 1859 (2018) 253–262 Available online 31 January 2018 0005-2728/ © 2018 Published by Elsevier B.V. T