Photosensitised Multiheme Cytochromes as Light‐Driven Molecular Wires and Resistors

Abstract

Multiheme cytochromes possess closely packed redox-active hemes arranged as chains spanning the tertiary structure. Here we describe five variants of a representative multiheme cytochrome engineered as biohybrid phototransducers for converting light into electricity. Each variant possesses a single Cys sulfhydryl group near a terminus of the heme chain, and this was efficiently labelled with a Ru (2,2'-bipyridine) photosensitiser. When irradiated in the presence of a sacrificial electron donor (SED) the proteins exhibited different types of behaviour. Certain proteins were rapidly and fully reduced. Other proteins were rapidly semi-reduced but resisted complete photoreduction. These findings reveal that photosensitised multiheme cytochromes can be engineered to act as resistors, with intrinsic regulation of light-driven electron accumulation, and also as molecular wires with essentially unhindered photoreduction. It is proposed that the observed behaviour arises from interplay between the site of electron injection and the distribution of heme reduction potentials along the heme chain.

Bespoke Biomolecular Wires for Transmembrane Electron Transfer: Spontaneous Assembly of a Functionalized Multiheme Electron Conduit

“…The thiol-reactive dye [Ru(4-bromomethyl-4methylbipyridine)(2,2 -bipyridine) 2 ](PF 6 ) 2 (Geren et al, 1991;Millett and Durham, 2002) has been successfully attached to cysteine residues on the surfaces of a number of redox proteins. This dye has a well-characterized luminescence that is sensitive to local environment (van Wonderen et al, 2018). Furthermore, the photoexcited dye is capable of injecting photoenergized electrons into multiheme cytochromes including S. oneidensis STC (van Wonderen et al, 2019) and of PpcA of Geobacter sulfurreducens (Kokhan et al, 2015).…”

“…Initial investigations took advantage of the Ru-dye attached to MtrC. The photoexcited triplet state of this dye, generated by absorption of photons at blue wavelengths, is a strong reductant (E m approximately -830 mV vs. SHE) with a lifetime of approximately 600 ns that is capable of transferring its photoenergized electron to a nearby protein cofactor (e.g., Geren et al, 1991;Millett and Durham, 2002;van Wonderen et al, 2018van Wonderen et al, , 2019. The photoenergized electron becomes trapped in the protein if the oxidized dye is reduced by a sacrificial electron donor and provided there are no sacrificial acceptors present.…”

“…The photoenergized electron becomes trapped in the protein if the oxidized dye is reduced by a sacrificial electron donor and provided there are no sacrificial acceptors present. For cytochromes with multiple hemes, such photoreduction is cumulative (van Wonderen et al, 2018) and readily quantified by changes in absorbance of the Soret band as illustrated for Ru-MtrC (e.g., Figure 4A). Initially, the protein is fully oxidized with all hemes in the Fe(III) state as indicated by the Soret band with maximum absorbance at 410 nm (Figure 4A, thick red line).…”

“…The Ru-dye-labeled protein is termed Ru-MtrC (Supplementary Figure 2). For LC-MS, performed as described in van Wonderen et al (2018), protein (typically >2 mg ml −1 ) was diluted with formic acid (0.1%, v/v) and acetonitrile (2%, v/v) prior to analysis.…”

Nanosecond heme-to-heme electron transfer rates in a multiheme cytochrome nanowire reported by a spectrally unique His/Met-ligated heme

“…Protein Preparation and Characterization. Ru-MtrC Met8, Ru-MtrC His8, and H561M MtrC were prepared using previously explained methods (53,54) and described in detail in SI Appendix. Potentiometric titration as described in ref.…”