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The fusion of hydrogenases and photosynthetic reaction centers (RCs) has proven to be a promising strategy for the production of sustainable biofuels. Type I (iron-sulfur-containing) RCs, acting as photosensitizers, are capable of promoting electrons to a redox state that can be exploited by hydrogenases for the reduction of protons to dihydrogen (H₂). While both [FeFe] and [NiFe] hydrogenases have been used successfully, they tend to be limited due to either O₂sensitivity, binding specificity, or H₂production rates. In this study, we fuse a peripheral (stromal) subunit of Photosystem I (PS I), PsaE, to an O₂-tolerant [FeFe] hydrogenase fromClostridium beijerinckiiusing a flexible [GGS]₄linker group (CbHydA1-PsaE). We demonstrate that theCbHydA1 chimera can be synthetically activated in vitro to show bidirectional activity and that it can be quantitatively bound to a PS I variant lacking the PsaE subunit. When illuminated in an anaerobic environment, the nanoconstruct generates H₂at a rate of 84.9 ± 3.1 µmol H₂mg_chl^–1h^–1. Further, when prepared and illuminated in the presence of O₂, the nanoconstruct retains the ability to generate H₂, though at a diminished rate of 2.2 ± 0.5 µmol H₂mg_chl^–1h^–1. This demonstrates not only that PsaE is a promising scaffold for PS I-based nanoconstructs, but the use of an O₂-tolerant [FeFe] hydrogenase opens the possibility for an in vivo H₂generating system that can function in the presence of O₂. 

Chlorophylls (Chl)s exist in a variety of flavors and are ubiquitous in both the energy and electron transfer processes of photosynthesis. The functions they perform often occur on the ultrafast (fs–ns) time scale and until recently, these have been difficult to measure in real time. Further, the complexity of the binding pockets and the resulting protein-matrix effects that alter the respective electronic properties have rendered theoretical modeling of these states difficult. Recent advances in experimental methodology, computational modeling, and emergence of new reaction center (RC) structures have renewed interest in these processes and allowed researchers to elucidate previously ambiguous functions of Chls and related pheophytins. This is complemented by a wealth of experimental data obtained from decades of prior research. Studying the electronic properties of Chl molecules has advanced our understanding of both the nature of the primary charge separation and subsequent electron transfer processes of RCs. In this review, we examine the structures of primary electron donors in Type I and Type II RCs in relation to the vast body of spectroscopic research that has been performed on them to date. Further, we present density functional theory calculations on each oxidized primary donor to study both their electronic properties and our ability to model experimental spectroscopic data. This allows us to directly compare the electronic properties of hetero- and homodimeric RCs.