The recently realized biochemical phenomenon of energy conservation through electron bifurcation provides biology with an elegant means to maximize utilization of metabolic energy. The mechanism of coordinated coupling of exergonic and endergonic oxidation-reduction reactions by a single enzyme complex has been elucidated through optical and paramagnetic spectroscopic studies revealing unprecedented features. Pairs of electrons are bifurcated over more than 1 volt of electrochemical potential by generating a low-potential, highly energetic, unstable flavin semiquinone and directing electron flow to an iron-sulfur cluster with a highly negative potential to overcome the barrier of the endergonic half reaction. The unprecedented range of thermodynamic driving force that is generated by flavin-based electron bifurcation accounts for unique chemical reactions that are catalyzed by these enzymes.

The biological reduction of dinitrogen (N) to ammonia (NH) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (E = -320 mV) coupled to reduction of flavodoxin semiquinone (E = -460 mV) and reduction of coenzyme Q (E = 10 mV). Knocking out fix genes rendered Δrnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.

Although a number of solar biohydrogen systems employing photosystem I (PSI) have been developed, few attain the electron transfer throughput of oxygenic photosynthesis. We have optimized a biological/organic nanoconstruct that directly tethers F B , the terminal þ þ 2 ferredoxin red is carried out in two separate photochemical half-reactions. Photosystem II (PSII) catalyzes the anodic half-cell reaction H 2 O þ plastoquinone-9 þ 2 hν → 1∕2 O 2 þ plastoquinol-9, while photosystem I (PSI) catalyzes the cathodic half-cell reaction cytochrome c 6ðredÞ þ ferredoxin ox þ 1 hν → cytochrome c 6ðoxÞ þ ferredoxin red . Visible photons provide the energy necessary to drive these otherwise thermodynamically unfavorable half-cell reactions to completion (1). Cyanobacteria evolve O 2 at a rate of approximately 400 μmol mg Chl −1 h −1 (2, 3) in a process limited by diffusiongoverned electron transfer steps (Fig. 1A), in particular the slow interaction of plastoquinol-9 with the cytochrome b 6 f complex (4). Once electrons leave PSI, diffusionally governed electron transfer steps constrain the rate of interaction of ferredoxin with other enzymes, including ferredoxin:NADP þ oxidoreductase. Were it possible to directly connect redox proteins through their redox centers, electrons could be vectored preferentially thereby eliminating any dependence on diffusional electron transfer (5, 6, 7). Here we report that by engineering a nanoconstruct in which both the electron donor (cytochrome c 6 ) and acceptor (here: ½FeFe-H 2 ase) are tethered to PSI in vitro, rate-limiting, diffusion-based electron transfer reactions are eliminated (Fig. 1B), resulting in electron transfer rates that exceed those of natural photosynthesis.The approach connects PSI to an ½FeFe-H 2 ase (8) using a molecular wire, which separates the [4Fe-4S] clusters of each enzyme by a defined distance (5). By introducing an exchangeable sulfhydryl ligand to the most solvent-exposed iron atom of PSI, the molecular wire can be attached by a ligand exchange mechanism. This is achieved by site-specific conversion of a ligating Cys residue (C13) of F B , the terminal [4Fe-4S] cluster, to a Gly (9-11) and by chemically rescuing the cluster with a small sulfhydrylcontaining molecule (11). Because ½FeFe-H 2 ases also contain [4Fe-4S] clusters, which constitute an electron transfer pathway between the surface of the enzyme and its catalytic site (12, 13), a similar strategy is used to introduce an exchangeable ligand at the distal [4Fe-4S] cluster (C97G). A tether that contains two sulfhydryl groups serves as a chemical rescue agent for both the F B cluster of PSI and the distal [4Fe-4S] cluster of ½FeFe-H 2 ase, thereby providing a pathway for electrons to quantum mechanically tunnel between the two proteins.

The generation of H(2) by the use of solar energy is a promising way to supply humankind's energy needs while simultaneously mitigating environmental concerns that arise due to climate change. The challenge is to find a way to connect a photochemical module that harnesses the sun's energy to a catalytic module that generates H(2) with high quantum yields and rates. In this review, we describe a technology that employs a "molecular wire" to connect a terminal [4Fe-4S] cluster of Photosystem I directly to a catalyst, which can be either a Pt nanoparticle or the distal [4Fe-4S] cluster of an [FeFe]- or [NiFe]-hydrogenase enzyme. The keys to connecting these two moieties are surface-located cysteine residues, which serve as ligands to Fe-S clusters and which can be changed through site-specific mutagenesis to glycine residues, and the use of a molecular wire terminated in sulfhydryl groups to connect the two modules. The sulfhydryl groups at the end of the molecular wire form a direct chemical linkage to a suitable catalyst or can chemically rescue a [4Fe-4S] cluster, thereby generating a strong coordination bond. Specifically, the molecular wire can connect the F(B) iron-sulfur cluster of Photosystem I either to a Pt nanoparticle or, by using the same type of genetic modification, to the differentiated iron atom of the distal [4Fe-4S].(Cys)(3)(Gly) cluster of hydrogenase. When electrons are supplied by a sacrificial donor, this technology forms the cathode of a photochemical half-cell that evolves H(2) when illuminated. If such a device were connected to the anode of a photochemical half-cell that oxidizes water, an in vitro solar energy converter could be realized that generates only O(2) and H(2) in the light. A similar methodology can be used to connect Photosystem I to other redox proteins that have surface-located [4Fe-4S] clusters. The controlled light-driven production of strong reductants by such systems can be used to produce other biofuels or to provide mechanistic insights into enzymes catalyzing multielectron, proton-coupled reactions.

Photosystem I (PS I) is a robust photosynthetic complex that adeptly captures photons to create a charge-separated state with a quantum efficiency that approaches 1.0. This charge-separated state is stable for approximately 100 ms, and the low-potential reductant that is produced is poised at a redox potential favorable for H2 evolution. PS I has been covalently linked to Pt and Au nanoparticle surfaces by 1,6-hexanedithiol which serves as a molecular wire to both connect PS I to the particles and transfer electrons from the terminal electron transfer cofactor of PS I, FB, to the nanoparticle. Illumination of these Photosystem I/molecular wire/nanoparticle bioconjugates is able to catalyze the reaction: 2H+ + 2e(-)--> H2. Transfer of the electrons from PS I to the nanoparticle through the molecular wire is not rate-limiting for H2 evolution. Supplying the system with more efficient donor-side electron donating species results in a 5-fold increase in the rate of H2 evolution.