Efficient nanomaterials for artificial photosynthesis require fast and robust unidirectional electron transfer (ET) from photosensitizers through charge-separation and accumulation units to redox-active catalytic sites. We explored the ultrafast time-scale limits of photo-induced charge transfer between a Ru(II)tris(bipyridine) derivative photosensitizer and PpcA, a 3-heme c-type cytochrome serving as a nanoscale biological wire. Four covalent attachment sites (K28C, K29C, K52C, and G53C) were engineered in PpcA enabling site-specific covalent labeling with expected donor-acceptor (DA) distances of 4–8 Å. X-ray scattering results demonstrated that mutations and chemical labeling did not disrupt the structure of the proteins. Time-resolved spectroscopy revealed three orders of magnitude difference in charge transfer rates for the systems with otherwise similar DA distances and the same number of covalent bonds separating donors and acceptors. All-atom molecular dynamics simulations provided additional insight into the structure-function requirements for ultrafast charge transfer and the requirement of van der Waals contact between aromatic atoms of photosensitizers and hemes in order to observe sub-nanosecond ET. This work demonstrates opportunities to utilize multi-heme c-cytochromes as frameworks for designing ultrafast light-driven ET into charge-accumulating biohybrid model systems, and ultimately for mimicking the photosynthetic paradigm of efficiently coupling ultrafast, light-driven electron transfer chemistry to multi-step catalysis within small, experimentally versatile photosynthetic biohybrid assemblies.

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C-type cytochromes play an important role in respiration of dissimilatory metal-reducing bacteria. They form extended conduits for charge transfer between the cellular metabolism and external electron acceptors such as particles of iron oxide, metal ions, and humic substances. Out of more than a hundred c-type cytochromes in Geobacter sulfurreducens, only a small fraction has been previously characterized. Here we present our results on expression and biophysical characterization of GSU0105, a novel 3-heme cytochrome, important for Fe(III) respiration in G. sulfurreducens. We successfully cloned the gene and achieved ~3 mg/L of culture GSU0105 expression in E.coli. Despite a similar size (71 amino acids) and the same number of c-type hemes to the members of the cytochrome (cyt) c7 family, multiple sequence alignment suggests that GSU0105 does not belong to the cyt c7 family. UV-Vis spectroscopy revealed typical c-type cytochrome spectral features, including a weak iron-sulfur charge transfer band suggesting that at least one heme is ligated with a methionine residue. Far UV circular dichroism studies demonstrate approximately 35% content of α-helices and β-sheets, each, as well as thermal aggregation occurring above 60C. A combination of SAXS and analytical size exclusion chromatography data shows that GSU0105 is monomeric in solution. Finally, affinity pull-down assays demonstrate high binding affinity to PpcD and weaker binding to the other members of the cyt c7 family.

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