John H. Golbeck

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components

Significance Bacterial nanowires from Shewanella oneidensis MR-1 were previously shown to be conductive under nonphysiological conditions. Intense debate still surrounds the molecular makeup, identity of the charge carriers, and cellular respiratory impact of bacterial nanowires. In this work, using in vivo fluorescence measurements, immunolabeling, and quantitative gene expression analysis, we demonstrate that S. oneidensis MR-1 nanowires are extensions of the outer membrane and periplasm, rather than pilin-based structures, as previously thought. We also demonstrate that the outer membrane multiheme cytochromes MtrC and OmcA localize to these membrane extensions, directly supporting one of the two models of electron transport through the nanowires; consistent with this, production of bacterial nanowires correlates with an increase in cellular reductase activity. Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic–abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

PsaE Is Required for in Vivo Cyclic Electron Flow around Photosystem I in the Cyanobacterium Synechococcus sp. PCC 7002

Electron transfer rates to P700+ have been determined in wild-type and three interposon mutants (psaE-, ndhF-, and psaE- ndhF-) of Synechococcus sp. PCC 7002. All three mutants grew significantly more slowly than wild type at low light intensities, and each failed to grow photoheterotrophically in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and a metabolizable carbon source. The kinetics of P700+ reduction were similar in the wild-type and mutant whole cells in the absence of DCMU. In the presence of DCMU, the P700+ reduction rate in the psaE mutant was significantly slower than in the wild type. In the presence of DCMU and potassium cyanide, added to inhibit the outflow of electrons through cytochrome oxidase, P700+ reduction rates increased for both the psaE- and ndhF- strains. The reduction rates for these two mutants were nonetheless slower than that observed for the wild-type strain. The further addition of methyl viologen caused the rate of P700+ reduction in the wild type to become as slow as that for the psaE mutant in the absence of methyl viologen. Given the ability of methyl viologen to intercept electrons from the acceptor side of photosystem I, this response reveals a lesion in cyclic electron flow in the psaE mutant. In the presence of DCMU, the rate of P700+ reduction in the psaE ndhF double mutant was very slow and nearly identical with that for the wild-type strain in the presence of 2,4-dibromo-3-methyl-6-isopropyl-p-benzoquinone, a condition under which physiological electron donation to P700+ should be completely inhibited. These results suggest that NdhF- and PsaE-dependent electron donation to P700+ occurs only via plastoquinone and/or cytochrome b6/f and indicate that there are three major electron sources for P700+ reduction in this cyanobacterium. We conclude that, although PsaE is not required for linear electron flow to NADP+, it is an essential component in the cyclic electron transport pathway around photosystem I.

Purification and properties of the intact P-700 and F x-containing Photosystem I core protein***

The intact Photosystem I core protein, containing the psaA and psaB polypeptides, and electron transfer components P-700 through FX, was isolated from cyanobacterial and higher plant Photosystem I complexes with chaotropic agents followed by sucrose density ultracentrifugation. The concentrations of NaClO4, NaSCN, NaI, NaBr or urea required for the functional removal of the 8.9 kDa, FA/FB polypeptide was shown to be inversely related to the strength of the chaotrope. The Photosystem I core protein, which was purified to homogeniety, contains 4 mol of acid-labile sulfide and has the following properties: (i) the FX-containing core consists of the 82 and 83 kDa reaction center polypeptides but is totally devoid of the low-molecular-mass polypeptides; (ii) methyl viologen and other bipyridilium dyes have the ability to accept electrons directly from FX; (iii) the difference spectrum of FX from 400 to 900 nm is characteristic of an iron-sulfur cluster; (iv) the midpoint potential of FX, determined optically at room temperature, is 60 mV more positive than in the control; (v) there is indication by ESR spectroscopy of low-temperature heterogeneity within FX; and (vi) the heterogeneity is seen by optical spectroscopy as inefficiency in low-temperature electron flow to FX. The constraints imposed by the amount of non-heme iron and labile sulfide in the Photosystem I core protein, the cysteine content of the psaA and psaB polypeptides, and the stoichiometry of high-molecular-mass polypeptides, cause us to re-examine the possibility that FX is a [4Fe-4S] rather than a [2Fe-2S] cluster ligated by homologous cysteine residues on the psaA and psaB heterodimer.

Recruitment of a Foreign Quinone into the A1 Site of Photosystem I I. GENETIC AND PHYSIOLOGICAL CHARACTERIZATION OF PHYLLOQUINONE BIOSYNTHETIC PATHWAY MUTANTS IN SYNECHOCYSTIS SP. PCC 6803

Genes encoding enzymes of the biosynthetic pathway leading to phylloquinone, the secondary electron acceptor of photosystem (PS) I, were identified inSynechocystis sp. PCC 6803 by comparison with genes encoding enzymes of the menaquinone biosynthetic pathway inEscherichia coli. Targeted inactivation of themenA and menB genes, which code for phytyl transferase and 1,4-dihydroxy-2-naphthoate synthase, respectively, prevented the synthesis of phylloquinone, thereby confirming the participation of these two gene products in the biosynthetic pathway. The menA and menB mutants grow photoautotrophically under low light conditions (20 μE m−2 s−1), with doubling times twice that of the wild type, but they are unable to grow under high light conditions (120 μE m−2 s−1). The menA andmenB mutants grow photoheterotrophically on media supplemented with glucose under low light conditions, with doubling times similar to that of the wild type, but they are unable to grow under high light conditions unless atrazine is present to inhibit PS II activity. The level of active PS II per cell in the menAand menB mutant strains is identical to that of the wild type, but the level of active PS I is about 50–60% that of the wild type as assayed by low temperature fluorescence, P700 photoactivity, and electron transfer rates. PS I complexes isolated from themenA and menB mutant strains contain the full complement of polypeptides, show photoreduction of FA and FB at 15 K, and support 82–84% of the wild type rate of electron transfer from cytochrome c 6 to flavodoxin. HPLC analyses show high levels of plastoquinone-9 in PS I complexes from the menA and menB mutants but not from the wild type. We propose that in the absence of phylloquinone, PS I recruits plastoquinone-9 into the A1site, where it functions as an efficient cofactor in electron transfer from A0 to the iron-sulfur clusters.

Photosystem I in Cyanobacteria

The Photosystem I (PS I) complex in cyanobacteria functions most typically as a light-driven, cytochrome c 6:ferredoxin oxidoreductase. The adaptability of cyanobacteria to conditions of nutrient availability allows cytochrome c 6 to be replaced by plastocyanin when copper is plentiful, and ferredoxin to be replaced by flavodoxin when iron is limiting. These changes, however, do not lead to any known alterations in the polypeptide composition of the membrane-bound PS I complex. This multiprotein complex incorporates all of the biochemical machinery required to produce efficient charge separation across the thylakoid membrane in a process that culminates in the conversion of a red photon to chemical free energy. The membrane-bound components which comprise the complex include an array of ∼ 110 antenna chlorophyll a molecules to provide a large optical cross-section to incoming photons, a series of inorganic and organic cofactors to carry out the acts of charge separation and charge stabilization, and a matrix of eleven polypeptides to provide ligands to the photoactive components. These components are arranged in a motif believed to be shared by all photochemical reaction centers: in PS I a chlorophyll (a) dimer serves as the primary electron donor; a chlorophyll (a) monomer serves as the primary electron acceptor; and a quinone (phylloquinone) serves as the intermediate electron acceptor. Other common features include the presence of a protein (hetero)dimer (PsaA and PsaB), which binds the antenna chlorophylls, the electron donor and acceptor chlorophylls, and the two quinone molecules. This shared photochemical motif is broken by the inter-polypeptide iron-sulfur cluster Fx, which occupies the same relative position as the non-heme iron in Type-II (quinone-type) reaction centers, but which is redox active in Type I (iron-sulfur type) reaction centers. The addition of two iron-sulfur clusters, FB and FA, located on a separate polypeptide, PsaC, provides a path for the electrons out of the membrane phase and to the stromal phase, allowing ferredoxin to be reduced with high quantum efficiency. The other PS I polypeptides serve ancillary roles in stabilizing PsaC and docking ferredoxin or flavodoxin (PsaD), in enhancing ferredoxin reduction and allowing for cyclic electron flow (PsaE), and in forming trimers of the PS I complex in the membrane and facilitating state transitions (PsaL). The functions of the remaining polypeptides, PsaF, PsaI, PsaJ, PsaK, and PsaM, are unclear; however it is increasingly unlikely that they participate directly in the primary processes of photochemical energy conversion.

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.

Assembly of photosystem I. I. Inactivation of the rubA gene encoding a membrane-associated rubredoxin in the cyanobacterium Synechococcus sp. PCC 7002 causes a loss of photosystem I activity.

A 4.4-kb HindIII fragment, encoding an unusual rubredoxin (denoted RubA), a homolog of theSynechocystis sp. PCC 6803 gene slr2034 andArabidopsis thaliana HCF136, and the psbEFLJoperon, was cloned from the cyanobacterium Synechococcussp. PCC 7002. Inactivation of the slr2034 homolog produced a mutant with no detectable phenotype and wild-type photosystem (PS) II levels. Inactivation of the rubA gene ofSynechococcus sp. PCC 7002 produced a mutant unable to grow photoautotrophically. RubA and PS I electron transport activity were completely absent in the mutant, although PS II activity was ∼80% of the wild-type level. RubA contains a domain of ∼50 amino acids with very high similarity to the rubredoxins of anaerobic bacteria and archaea, but it also contains a region of about 50 amino acids that is predicted to form a flexible hinge and a transmembrane α-helix at its C terminus. Overproduction of the water-soluble rubredoxin domain inEscherichia coli led to a product with the absorption and EPR spectra of typical rubredoxins. RubA was present in thylakoid but not plasma membranes of cyanobacteria and in chloroplast thylakoids isolated from spinach and Chlamydomonas reinhardtii. Fractionation studies suggest that RubA might transiently associate with PS I monomers, but no evidence for an association with PS I trimers or PS II was observed. PS I levels were significantly lower than in the wild type (∼40%), but trimeric PS I complexes could be isolated from the rubA mutant. These PS I complexes completely lacked the stromal subunits PsaC, PsaD, and PsaE but contained all membrane-intrinsic subunits. The three missing proteins could be detected immunologically in whole cells, but their levels were greatly reduced, and degradation products were also detected. Our results indicate that RubA plays a specific role in the biogenesis of PS I.

Iron–sulfur clusters in type I reaction centers

Type I reaction centers (RCs) are multisubunit chlorophyll-protein complexes that function in photosynthetic organisms to convert photons to Gibbs free energy. The unique feature of Type I RCs is the presence of iron-sulfur clusters as electron transfer cofactors. Photosystem I (PS I) of oxygenic phototrophs is the best-studied Type I RC. It is comprised of an interpolypeptide [4Fe-4S] cluster, F(X), that bridges the PsaA and PsaB subunits, and two terminal [4Fe-4S] clusters, F(A) and F(B), that are bound to the PsaC subunit. In this review, we provide an update on the structure and function of the bound iron-sulfur clusters in Type I RCs. The first new development in this area is the identification of F(A) as the cluster proximal to F(X) and the resolution of the electron transfer sequence as F(X)-->F(A)-->F(B)-->soluble ferredoxin. The second new development is the determination of the three-dimensional NMR solution structure of unbound PsaC and localization of the equal- and mixed-valence pairs in F(A)(-) and F(B)(-). We provide a survey of the EPR properties and spectra of the iron-sulfur clusters in Type I RCs of cyanobacteria, green sulfur bacteria, and heliobacteria, and we summarize new information about the kinetics of back-reactions involving the iron-sulfur clusters.

Photosystem I charge separation in the absence of centers A and B. I. Optical characterization of center ‘A2’ and evidence for its association with a 64-kDa peptide

Abstract The flash-induced absorption transient at 698 nm in a Photosystem I subchloroplast particle showed the following characteristics after addition of 0.25–2.0% lithium dodecyl sulfate (LDS). (i) The 30-ms transient corresponding to the P-700 + P-430 − backreaction was replaced by a 1.2-ms transient. (ii) The amplitude of the transient did not change immediately after LDS addition, but decayed with a half-life of 10 min at pH 8.5. (iii) Methyl viologen had no effect on the magnitude or kinetics of the transient, indicating that it cannot accept an electron from this component. (iv) The difference spectrum of the transient from 400 nm to 500 nm was characteristic of an iron-sulfur protein. (v) The transient followed first-order Arrhenius behavior between 298 K and 225 K with an activation energy of 13.3 kJ/mol; between 225 K and 77 K, the 85-ms half-time remained temperature-invariant. These properties suggest that the LDS-induced absorption transient corresponds to the P-700 + A − 2 change recombination seen in the absence of a reduced electron-acceptor system. In the presence of LDS, the reaction-center complex was dissociated, allowing removal of the smaller peptides from the 64-kDa P-700-containing protein. With prolonged incubation, the iron-sulfur clusters were destroyed through conversion of the labile sulfide to zero-valence sulfur. About 35% of the zero-valence sulfur was found associated with the 64-kDa protein under conditions that allowed separation of the small peptides. We interpret the long lifetime of the P-700 + A − 2 transient after LDS addition and the association of zero-valence sulfur with a 64-kDa protein to indicate that A 2 is closely associated with, and perhaps integral with, the P-700-containing protein.