Carolyn E. Lubner

Solar hydrogen-producing bionanodevice outperforms natural photosynthesis

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 FB, the terminal [4Fe-4S] cluster of PSI from Synechococcus sp. PCC 7002, to the distal [4Fe-4S] cluster of the [FeFe]-hydrogenase (H2ase) from Clostridium acetobutylicum. On illumination, the PSI–[FeFe]-H2ase nanoconstruct evolves H2 at a rate of 2,200 ± 460 μmol mg chlorophyll-1 h-1, which is equivalent to 105 ± 22 e-PSI-1 s-1. Cyanobacteria evolve O2 at a rate of approximately 400 μmol mg chlorophyll-1 h-1, which is equivalent to 47 e-PSI-1 s-1, given a PSI to photosystem II ratio of 1.8. The greater than twofold electron throughput by this hybrid biological/organic nanoconstruct over in vivo oxygenic photosynthesis validates the concept of tethering proteins through their redox cofactors to overcome diffusion-based rate limitations on electron transfer.

Photosystem I/molecular wire/metal nanoparticle bioconjugates for the photocatalytic production of H2.

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.

Wiring an [FeFe]-hydrogenase with photosystem I for light-induced hydrogen production.

A molecular wire is used to connect two proteins through their physiologically relevant redox cofactors to facilitate direct electron transfer. Photosystem I (PS I) and an [FeFe]-hydrogenase (H(2)ase) serve as the test bed for this new technology. By tethering a photosensitizer with a hydrogen-evolving catalyst, attached by Fe-S coordination bonds between the F(B) iron-sulfur cluster of PS I and the distal iron-sulfur cluster of H(2)ase, we assayed electron transfer between the two components via light-induced hydrogen generation. These hydrogen-producing nanoconstructs self-assemble when the PS I variant, the H(2)ase variant, and the molecular wire are combined.

A novel photosynthetic strategy for adaptation to low-iron aquatic environments

Iron (Fe) availability is a major limiting factor for primary production in aquatic environments. Cyanobacteria respond to Fe deficiency by derepressing the isiAB operon, which encodes the antenna protein IsiA and flavodoxin. At nanomolar Fe concentrations, a PSI-IsiA supercomplex forms, comprising a PSI trimer encircled by two complete IsiA rings. This PSI-IsiA supercomplex is the largest photosynthetic membrane protein complex yet isolated. This study presents a detailed characterization of this complex using transmission electron microscopy and ultrafast fluorescence spectroscopy. Excitation trapping and electron transfer are highly efficient, allowing cyanobacteria to avoid oxidative stress. This mechanism may be a major factor used by cyanobacteria to successfully adapt to modern low-Fe environments.

Mechanistic insights into energy conservation by flavin-based electron bifurcation

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.

Two-dimensional protein crystals for solar energy conversion.

Two-dimensional photosynthetic protein crystals provide a high density of aligned reaction centers. We reconstitute the robust light harvesting protein Photosystem I into a 2D crystal with lipids and integrate the crystals into a photo-electrochemical device. A 4-fold photocurrent enhancement is measured by incorporating conjugated oligoelectrolytes to form a supporting conductive bilayer in the device which produces a high photocurrent of ∼600 μA per mg PSI deposited.

The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis

The biological reduction of dinitrogen (N2) to ammonia (NH3) 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 (Em = -320 mV) coupled to reduction of flavodoxin semiquinone (Em = -460 mV) and reduction of coenzyme Q (Em = 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.

Equilibrium and ultrafast kinetic studies manipulating electron transfer: A short-lived flavin semiquinone is not sufficient for electron bifurcation

Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential (i.e. intermediate reducing power), and each electron is passed to a different acceptor, one with lower and the other with higher reducing power, leading to “bifurcation.” It is believed that a strongly reducing semiquinone species is essential for this process, and it is expected that this species should be kinetically short-lived. We now demonstrate that the presence of a short-lived anionic flavin semiquinone (ASQ) is not sufficient to infer the existence of bifurcating activity, although such a species may be necessary for the process. We have used transient absorption spectroscopy to compare the rates and mechanisms of decay of ASQ generated photochemically in bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase and the non-bifurcating flavoproteins nitroreductase, NADH oxidase, and flavodoxin. We found that different mechanisms dominate ASQ decay in the different protein environments, producing lifetimes ranging over 2 orders of magnitude. Capacity for electron transfer among redox cofactors versus charge recombination with nearby donors can explain the range of ASQ lifetimes that we observe. Our results support a model wherein efficient electron propagation can explain the short lifetime of the ASQ of bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase I and can be an indication of capacity for electron bifurcation.

Wiring photosystem I for electron transfer to a tethered redox dye

We have recently reported the assembly of biological/organic hybrid nanoconstructs that generate H2 in the light (Grimme et al., Dalton Trans., 2009, 10106, Lubner et al., Biochemistry, 2010, 49, 10264). In these constructs, electrons are transferred directly from a photochemical module, Photosystem I (PS I), to a catalytic module, either a Pt nanoparticle (NP) or an [FeFe]-hydrogenase (H2ase), through the use of a covalently attached molecular wire. In neither case are any spectroscopic changes visible that would allow electron transfer to be monitored between the photochemical and catalytic modules. In this study, the catalytic module was replaced with an organic cofactor consisting of 1-(3-thiopropyl)-1′-(methyl)-4,4′-bipyridinium chloride that allowed electron transfer to be measured to a spectroscopically observable marker. EPR and optical spectroscopy showed that the tethered redox cofactor was attached to PS I through the FB cluster of PsaC. Under steady-state illumination, the rate of reduction of the 4,4′-bipyridinium cofactor was comparable to the rate of H2 evolution observed for the PS I—molecular wire—Pt-NP and PS I—molecular wire—[FeFe]-H2ase nanoconstructs. These observations provide proof-of-concept for incorporating a redox cofactor in the molecular wire, thereby setting the stage for monitoring the rate and yield of electron transfer between PS I and the tethered [FeFe]-H2ase.

Reduction Potentials of [FeFe]-Hydrogenase Accessory Iron–Sulfur Clusters Provide Insights into the Energetics of Proton Reduction Catalysis

An [FeFe]-hydrogenase from Clostridium pasteurianum, CpI, is a model system for biological H2 activation. In addition to the catalytic H-cluster, CpI contains four accessory iron-sulfur [FeS] clusters in a branched series that transfer electrons to and from the active site. In this work, potentiometric titrations have been employed in combination with electron paramagnetic resonance (EPR) spectroscopy at defined electrochemical potentials to gain insights into the role of the accessory clusters in catalysis. EPR spectra collected over a range of potentials were deconvoluted into individual components attributable to the accessory [FeS] clusters and the active site H-cluster, and reduction potentials for each cluster were determined. The data suggest a large degree of magnetic coupling between the clusters. The distal [4Fe-4S] cluster is shown to have a lower reduction potential (∼ < -450 mV) than the other clusters, and molecular docking experiments indicate that the physiological electron donor, ferredoxin (Fd), most favorably interacts with this cluster. The low reduction potential of the distal [4Fe-4S] cluster thermodynamically restricts the Fdox/Fdred ratio at which CpI can operate, consistent with the role of CpI in recycling Fdred that accumulates during fermentation. Subsequent electron transfer through the additional accessory [FeS] clusters to the H-cluster is thermodynamically favorable.