Nitrous oxide (N2O) is a potent greenhouse and ozone-reactive gas for which emissions are growing rapidly due to increasingly intensive agriculture. Synthetic catalysts for N2O decomposition typically contain precious metals and/or operate at elevated temperatures driving a desire for more sustainable alternatives. Here we demonstrate self-assembly of liposomal microreactors enabling catalytic reduction of N2O to the climate neutral product N-2. Photoexcitation of graphitic N-doped carbon dots delivers electrons to encapsulated N2O Reductase enzymes via a lipid-soluble biomolecular wire provided by the MtrCAB protein complex. Within the microreactor, electron transfer from MtrCAB to N2O Reductase is facilitated by the general redox mediator methyl viologen. The liposomal microreactors use only earth-abundant elements to catalyze N2O removal in ambient, aqueous conditions.

Significance Multiheme cytochromes have been identified as essential proteins for electron exchange between bacterial enzymes and redox substrates outside of the cell. In microbiology, these proteins contribute to efficient energy storage and conversion. For biotechnology, multiheme cytochromes contribute to the production of green fuels and electricity. Furthermore, these proteins inspire the design of molecular-scale electronic devices. Here, we report exceptionally high rates of heme-to-heme electron transfer in a multiheme cytochrome. We expect similarly high rates, among the highest reported for ground-state electron transfer in biology, in other multiheme cytochromes as the close-packed hemes adopt similar configurations despite very different amino acid sequences and protein folds.

Shewanella oneidensis exchanges electrons between cellular metabolism and external redox partners in a process that attracts much attention for production of green electricity (microbial fuel cells) and chemicals (microbial electrosynthesis). A critical component of this pathway is the outer membrane spanning MTR complex, a biomolecular wire formed of the MtrA, MtrB, and MtrC proteins. MtrA and MtrC are decaheme cytochromes that form a chain of close-packed hemes to define an electron transfer pathway of 185 Å. MtrA is wrapped inside MtrB for solubility across the outer membrane lipid bilayer; MtrC sits outside the cell for electron exchange with external redox partners. Here, we demonstrate tight and spontaneous in vitro association of MtrAB with separately purified MtrC. The resulting complex is comparable with the MTR complex naturally assembled by Shewanella in terms of both its structure and rates of electron transfer across a lipid bilayer. Our findings reveal the potential for building bespoke electron conduits where MtrAB combines with chemically modified MtrC, in this case, labeled with a Ru-dye that enables light-triggered electron injection into the MtrC heme chain.

A growing number of bacterial species are known to move electrons across their cell envelopes. Naturally this occurs in support of energy conservation and carbon-fixation. For biotechnology it allows electron exchange between bacteria and electrodes in microbial fuel cells and during microbial electrosynthesis. In this contextRhodopseudomonas palustrisTIE-1 is of much interest. These bacteria respond to light by taking electrons from their external environment, including electrodes, to drive CO2-fixation. The PioA cytochrome, that spans the bacterial outer membrane, is essential for this electron transfer and yet little is known about its structure and electron transfer properties. Here we reveal the tenc-type hemes of PioA are redox active across the window +250 to -400 mV versus Standard Hydrogen Electrode and that the hemes with most positive reduction potentials have His/Met and His/H2O ligation. These chemical and redox properties distinguish PioA from the more widely studied family of MtrA outer membrane decaheme cytochromes with ten His/His ligated hemes. We predict a structure for PioA in which the hemes form a chain spanning the longest dimension of the protein, from Heme 1 to Heme 10. Hemes 2, 3 and 7 are identified as those most likely to have His/Met and/or His/H2O ligation. Sequence analysis suggests His/Met ligation of Heme 2 and/or 7 is a defining feature of decaheme PioA homologs from over 30 different bacterial genera. His/Met ligation of Heme 3 appears to be less common and primarily associated with PioA homologs from purple non-sulphur bacteria belonging to the alphaproteobacteria class.

Jenny Zhang opened the discussion of the paper by Shelley Minteer: Was the vision of this study to produce salinity adapted microorganisms in the lab and then release them into the environment? Or was it mainly to try to understand how salinity adaptation can occur in the natural environment, using RNA sequencing to facilitate this? Shelley Minteer replied: A comprehensive answer is that we are interested in both aspects. Speci  cally, our vision is that a better understanding of the salinity adaptation strategies will pose the basis to develop photo-bioelectrochemical systems capable of stable and long-term operation in saline environments. Furthermore, elucidating bacterial metabolism and their in  uence on photo-bioelectrocatalysis will not only allow for more robust and stable devices, but also for the better correlation of current response and changes taking place in the environment. Accordingly, we consider that the deeper understanding of biological processes related to the metabolism of electroactive microorganisms is critical for their successful application in the environment. You mentioned the of bio  lms. Is this a stress response to the pH of the medium the organisms exposed

Multiheme cytochromes attract much attention for their electron transport properties. These proteins conduct electrons across bacterial cell walls and along extracellular filaments and when purified can serve as bionanoelectronic junctions. Thus, it is important and necessary to identify and understand the factors governing electron transfer in this family of proteins. To this end we have used ultrafast transient absorbance spectroscopy, to define heme-heme electron transfer dynamics in the representative multiheme cytochrome STC from Shewanella oneidensis in aqueous solution. STC was photosensitized by site-selective labeling with a Ru(II)(bipyridine)3 dye and the dynamics of light-driven electron transfer described by a kinetic model corroborated by molecular dynamics simulation and density functional theory calculations. With the dye attached adjacent to STC Heme IV, a rate constant of 87 × 106 s-1 was resolved for Heme IV → Heme III electron transfer. With the dye attached adjacent to STC Heme I, at the opposite terminus of the tetraheme chain, a rate constant of 125 × 106 s-1 was defined for Heme I → Heme II electron transfer. These rates are an order of magnitude faster than previously computed values for unlabeled STC. The Heme III/IV and I/II pairs exemplify the T-shaped heme packing arrangement, prevalent in multiheme cytochromes, whereby the adjacent porphyrin rings lie at 90° with edge-edge (Fe-Fe) distances of ∼6 (11) Å. The results are significant in demonstrating the opportunities for pump-probe spectroscopies to resolve interheme electron transfer in Ru-labeled multiheme cytochromes.

The high quantum efficiency of natural photosynthesis has inspired chemists for solar fuel synthesis. In photosynthesis, charge recombination in photosystems is minimized by efficient charge separation across the thylakoid membrane. Building on our previous bioelectrochemical studies of electron transfer between a light-harvesting nanoparticle (LHNP) and the decahaem subunit MtrC, we demonstrate photoinduced electron transfer through the full transmembrane MtrCAB complex in liposome membranes. Successful photoelectron transfer is demonstrated by the decomposition of a redox dye, Reactive Red 120 (RR120), encapsulated in MtrCAB proteoliposomes. The photoreduction rates are found to be dependent on the identity of the external LHNPs, specifically, dye-sensitized TiO2, amorphous carbon dots (a-CD) and graphitic carbon dots with core nitrogen doping (g-N-CDs). Agglomeration or aggregation of TiO2 NPs likely reduces the kinetics of RR120 reductive decomposition. In contrast, with the dispersed a-CD and g-N-CDs, the kinetics of the RR120 reductive decomposition are observed to be faster with the MtrCAB proteoliposomes and we propose that this is due to enhancement in the charge-separated state. Thus, we show a proof-of-concept for using MtrCAB as a lipid membrane-spanning building block for compartmentalised photocatalysis that mimics photosynthesis. Future work is focused on incorporation of fuel generating redox catalysts in the MtrCAB proteoliposome lumen.

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-II(2,2-bipyridine)(3) 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.

Certain bacterial species have a natural ability to exchange electrons with extracellular redox partners. This behavior allows coupling of catalytic transformations inside bacteria to complementary redox transformations of catalysts and electrodes outside the cell. Electricity generation can be coupled to waste-water remediation. Industrially relevant oxidation reactions can proceed exclusively when electrons are released to anodes. Reduced products such as fuels can be generated when electrons are provided from (photo)cathodes. Rational development of these opportunities and inspiration for novel technologies is underpinned by resolution at the molecular level of pathways supporting electron exchange across bacterial cell envelopes. This chapter describes methods for purification, engineering, and in vitro characterization of proteins providing the primary route for electron transport across the outer membrane lipid bilayer of Shewanella oneidensis MR-1, a well-described electrogenic bacterium and chassis organism for related biotechnologies.