Biohybrid technologies like semiartificial photosynthesis are attracting increased attention, as they enable the combination of highly efficient synthetic light-harvesters with the self-healing and outstanding performance of biocatalysis. However, such systems are intrinsically complex, with multiple interacting components. Herein, we explore a whole-cell photocatalytic system for hydrogen (H 2 ) gas production as a model system for semiartificial photosynthesis. The employed whole-cell photocatalytic system is based on Escherichia coli cells heterologously expressing a highly efficient, but oxygen-sensitive, [FeFe] hydrogenase. The system is driven by the organic photosensitizer eosin Y under broad-spectrum white light illumination. The direct involvement of the [FeFe] hydrogenase in the catalytic reaction is verified spectroscopically. We also observe that E. coli provides protection against O 2 damage, underscoring the suitability of this host organism for oxygen-sensitive enzymes in the development of (photo) catalytic biohybrid systems. Moreover, the study shows how factorial experimental design combined with analysis of variance (ANOVA) can be employed to identify relevant variables, as well as their interconnectivity, on both overall catalytic performance and O 2 tolerance.

Both photo-and biocatalysis are well-established and intensively studied. The combination of these two approaches is also an emerging research field, commonly referred to as semi-artificial photosynthesis. Semi-artificial photosynthesis aims at combining highly efficient synthetic light harvesters with the self-healing and potent catalytic properties of biocatalysis. In this study, a semi-artificial photocatalytic system featuring Escherichia coli bacteria, which heterologously express the [FeFe] hydrogenase enzyme HydA1 from green algae, is employed as a hydrogen gas production catalyst. To probe the influence of photochemistry on overall system performance, the E. coli whole-cell catalyst is combined with two different photosensitizers and redox mediators. The addition of a redox mediator greatly improves the rates and longevity of the photocatalytic system, as reflected in increases of both the turn-over number (0.777 vs 10.9 μmol H 2 mL −1 OD 600 −1) and the turn-over frequency (175 vs 334 μmol H 2 mL −1 h −1 OD 600 −1). The redox mediator is found to both protect from photobleaching and enable electron transport to the hydrogenase from an extracellular photosensitizer. However, E. coli cells are strongly affected by the photocatalytic system, leading to a decrease in cell integrity and cell viability, possibly due to toxic decomposition products formed during the photocatalytic process. We furthermore employed steady-state and transient absorption spectroscopy to investigate solution potentials and the kinetics of electron transfer processes between the sacrificial electron donor, photosensitizer, redox mediator, and the [FeFe] hydrogenase as the final electron acceptor. These results allowed us to rationalize the different activities observed in photocatalytic assays and offer a better understanding of the factors that influence the photocatalytic performance of E. coli-based whole-cell systems.

In this study, we report two dinuclear Ru(II) complexes C1 and C2 and compare them to their mononuclear analogues Ref1 and Ref2. The dinuclear species exhibit a much stronger absorption,...