Contrasts between oxygenic and anoxygenic photoreduction of ferredoxin: Incompatibilities with prevailing concepts of photosynthetic electron transport

An investigation by paramagnetic resonance spectroscopy of the photoreduction of ferredoxin, oxygenically by water and anoxygenically by a direct electron donor to photosystem I, led to the unexpected findings that different reductive mechanisms may be involved. Ferredoxin photoreduced by water was not reoxidized in the light under aerobic conditions and, under anaerobic conditions, it was remarkably resistant to reoxidation in the dark. By contrast, ferredoxin photoreduced by a donor to photosystem I was readily reoxidized in the light by air and, under anaerobic conditions, by exposure to darkness. Furthermore, when electron transport linking photosystems I and II was inhibited by a plastoquinone antagonist, ferredoxin was photoreduced by water with no evidence for an accompanying photoreduction of the more electronegative bound iron-sulfur centers in chloroplasts. These findings are at variance with the now prevalent concepts of photosynthetic electron transport.     

Bacterial Na+-translocating ferredoxin:NAD+ oxidoreductase

The anaerobic acetogenic bacterium Acetobacterium woodii carries out a unique type of Na(+)-motive, anaerobic respiration with caffeate as electron acceptor, termed "caffeate respiration." Central, and so far the only identified membrane-bound reaction in this respiration pathway, is a ferredoxin:NAD(+) oxidoreductase (Fno) activity. Here we show that inverted membrane vesicles of A. woodii couple electron transfer from reduced ferredoxin to NAD(+) with the transport of Na(+) from the outside into the lumen of the vesicles. Na(+) transport was electrogenic, and accumulation was inhibited by sodium ionophores but not protonophores, demonstrating a direct coupling of Fno activity to Na(+) transport. Results from inhibitor studies are consistent with the hypothesis that Fno activity coupled to Na(+) translocation is catalyzed by the Rnf complex, a membrane-bound, iron-sulfur and flavin-containing electron transport complex encoded by many bacterial and some archaeal genomes. Fno is a unique type of primary Na(+) pump and represents an early evolutionary mechanism of energy conservation that expands the redox range known to support life. In addition, it explains the lifestyle of many anaerobic bacteria and gives a mechanistic explanation for the enigma of the energetic driving force for the endergonic reduction of ferredoxin with NADH plus H(+) as reductant in a number of aerobic bacteria.     

Reconstruction of the chloroplast noncyclic electron transport pathway from water to NADP with three integral protein complexes

Reconstruction of photosynthetic noncyclic electron transport from water to NADP has been accomplished by using three integral protein complexes isolated from chloroplast thylakoid membranes: photosystems I and II and the cytochrome b₆-f complex. This system shows an absolute dependence on the presence of all three protein complexes for NADP reduction, in addition to plastocyanin, ferredoxin, and ferredoxin-NADP reductase. The reconstructed system was found to be sensitive to low concentrations of known inhibitors of noncyclic electron transport. Depletion of the Rieske iron-sulfur center and bound plastoquinone from the cytochrome b₆-f complex resulted in an inhibition of the photoreduction of NADP.     

Primary Reactions of Photosynthesis: Photoreduction of a Bound Chloroplast Ferredoxin at Low Temperature as Detected by EPR Spectroscopy

An electron paramagnetic resonance signal was observed at 25 degrees K in whole spinach chloroplasts after illumination at 77 degrees K. The light-induced epr spectrum had g-values (g(x) = 1.86, g(y) = 1.94, g(z) = 2.05) and a temperature dependence that were characteristic of the reduced state of a plant-type ferredoxin. The light-induced epr spectrum was also observed in broken spinach chloroplasts from which soluble ferredoxin was removed. Chemical analyses showed that both whole and broken spinach chloroplasts contained amounts of nonheme iron and "acid-labile sulfide" consistent with the presence of a bound iron-sulfur protein, at a level of about one molecule per 75 chlorophyll molecules. These results support the conclusion that chloroplasts contain a bound ferredoxin that may serve as a primary low-potential electron acceptor in photosynthesis.     

THE ELECTRON TRANSPORT SYSTEM IN NITROGEN FIXATION BY Azotobacter. II. ISOLATION AND FUNCTION OF A NEW TYPE OF FERREDOXIN

A new type of ferredoxin was isolated from Azotobacter vinelandii cells. The protein was able to replace the native chloroplast ferredoxin in the photoreduction of nicotinamide adenine dinucleotide phosphate (NADP) and functioned as a reductant for the Azotobacter nitrogenase.     

Photoreduction of ferredoxin with various electron donors: Support for the Z scheme of photosynthetic electron transport

The currently accepted scheme for photosynthetic electron flow from water to ferredoxin (and subsequently to NADP), known as the Z scheme, envisions a linear electron flow that requires two photosystems joined by several electron carriers. The observation that the extent of photoreduction of ferredoxin depends on whether electrons are provided by water (a donor to photosystem II) or by artificial electron donors to photosystem I led Arnon et al. [Arnon, D. I., Tsujimoto, H. Y. & Tang, G. M.-S. (1980) Proc. Natl. Acad. Sei. USA 77, 2676-2680] to question the validity of the Z scheme for photosynthetic electron transport. Our results show that this difference is not due to any inherent difference in electron transport but to the fact that when electron donors are added to chloroplasts they oxidize (in their oxidized state) reduced ferredoxin. Different electron donors oxidize reduced ferredoxin to a different extent; dehydroascorbate is a more potent oxidant than dithiothreitol. Dichlorophenol-indophenol is also a potent oxidant of reduced ferredoxin. The rate of NADP reduction and the K_m for NADP with various electron donors reflect the oxidative capacities of the electron donors and mediators. These results also explain the fact that NADP reduction with electron donors to photosystem I is less than that with water, despite the fact that electron flow from donor to artificial dyes can proceed at high rates.     

Photoreduction of NADP+ by isolated reaction centers of photosystem II: requirement for plastocyanin.

The carrier of photosynthetically generated reducing power is the iron-sulfur protein ferredoxin, which provides directly, or via NADP+, reducing equivalents needed for CO2 assimilation and other metabolic reactions in the cell. It is now widely held that, in oxygenic photosynthesis, the generation of reduced ferredoxin-NADP+ requires the collaboration in series of two photosystems: photosystem II (PSII), which energizes electrons to an intermediate reducing potential and transfers them to photosystem I (PSI), which in turn is solely competent to energize electrons to the strong reducing potential required for the reduction of ferredoxin-NADP+ (the Z scheme). This investigation tested the premise of an alternative scheme, which envisions that PSII, without the involvement of PSI, is also capable of photoreducing ferredoxin-NADP+. We report here unexpected findings consistent with the alternative scheme. Isolated PSII reaction centers (completely free of PSI components), when supplemented with ferredoxin, ferredoxin-NADP+ oxidoreductase, and a PSII electron donor,1,5-diphenylcarbazide, gave a significant photoreduction of NADP+. A striking feature of this electron transfer from a PSII donor to the perceived terminal acceptor of PSI was its total dependence on catalytic quantities of plastocyanin, a copper-containing electron-transport protein hitherto known only as an electron donor to PSI.     

Proton transport in photooxidation of water: A new perspective on photosynthesis

The currently prevalent concept of the generation of photosynthetic reducing power in oxygen-evolving cells envisions a linear (noncyclic) electron flow from water to ferredoxin (and thence to NADP⁺) that requires the collaboration of photosystems I and II (PSI and PSII) joined by plastoquinone and other electron carriers (the Z scheme). The essence of the Z scheme is that only PSI can reduce ferredoxin—i.e., that, after being energized to an intermediate reducing potential by PSII, electrons from water are transported via plastoquinone to PSI which energizes the electrons to their ultimate reducing potential adequate for the reduction of ferredoxin. Basic to the Z scheme is the function of plastoquinone as the obligatory link in electron transport from PSII to PSI. However, we have found that, when plastoquinone function was inhibited, ferredoxin was photoreduced by water without the collaboration of PSI. We now report evidence for an important function of plastoquinone in the translocation of protons liberated inside the thylakoid membrane by photooxidation of water. When the oxygenic photoreduction (i.e., by water) of ferredoxin was blocked by plastoquinone inhibitors, dibromothymoquinone or dinitrophenol ether of iodonitrothymol, the photoreduction of ferredoxin was restored by each of four chemically diverse uncouplers, similar only in their ability to facilitate proton movement across membranes. Similar results were obtained for the oxygenic reduction of NADP⁺. Our results suggest that the light-induced electron flow from water cannot be maintained unless the simultaneously liberated protons are removed from inside the membrane via plastoquinone. The new evidence is embodied in a concept of an oxygenic photosystem for photosynthetic electron and proton transport, which we propose as an alternative to the Z scheme, to account for photoreduction of ferredoxin-NADP⁺ by water and the coupled oxygenic (formerly noncyclic) ATP formation without involving PSI. The role of the anoxygenic photosystem (formerly called PSI) is ATP formation by cyclic photophosphorylation.     

Photosynthetic electron partitioning between [FeFe]-hydrogenase and ferredoxin:NADP+-oxidoreductase (FNR) enzymes in vitro

Photosynthetic water splitting, coupled to hydrogenase-catalyzed hydrogen production, is considered a promising clean, renewable source of energy. It is widely accepted that the oxygen sensitivity of hydrogen production, combined with competition between hydrogenases and NADPH-dependent carbon dioxide fixation are the main limitations for its commercialization. Here we provide evidence that, under the anaerobic conditions that support hydrogen production, there is a significant loss of photosynthetic electrons toward NADPH production in vitro. To elucidate the basis for competition, we bioengineered a ferredoxin-hydrogenase fusion and characterized hydrogen production kinetics in the presence of Fd, ferredoxin:NADP(+)-oxidoreductase (FNR), and NADP(+). Replacing the hydrogenase with a ferredoxin-hydrogenase fusion switched the bias of electron transfer from FNR to hydrogenase and resulted in an increased rate of hydrogen photoproduction. These results suggest a new direction for improvement of biohydrogen production and a means to further resolve the mechanisms that control partitioning of photosynthetic electron transport.     

Characterization of the iron-sulfur centers in succinate dehydrogenase.

Two techniques have been applied to the determination of the number and type (2-Fe, 4-Fe) of iron-sulfur centers in the iron-sulfur flavoprotein succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.1]. One procedure uses p-CF3C6H4SH as an extrusion reagent and Fourier transform 19F nuclear magentic resonance as the method of detection and quantitation of extruded cores of these centers in the form of [Fe2S2(SRF)4]2- and [Fe4S4(SRF)4]2- (RF = p-C6H4CF3). The second procedure, interprotein core transfer, involves thiol displacement of iron-sulfur cores followed by specific core transfer to the apoproteins of Bacillus polymyxa ferredoxin and adrenodoxin. Detection and quantitation are accomplished by electron paramagnetic resonance of reduced proteins at low temperatures. Both procedures clearly show that succinate dehydrogenase contains two dimeric (Fe2S2) and one tetrameric (Fe4S4) centers per mole of histidyl flavin, accounting for all eight nonheme iron and eight labile sulfur atoms found by chemical analysis. These results remove uncertainties created by the less than stoichiometric amounts of binuclear centers detected by electron paramagnetic resonance after dithionite reduction and provide secure characterization of the iron-sulfur centers in this enzyme.     

Stabilization of electron spin resonance probes for photosynthesis studies.

The major obstacle to the study of functional/structural interrelationships of spinach chloroplasts by using spin labels has been the rapid loss of the electron paramagnetic resonance (EPR) signals upon illumination with visible light. The present study demonstrates that the addition of ferredoxin and NADP+ in the presence of N-tris(hydroxymethyl)methylglycine (Tricine) buffer at pH 7.1 or higher mitigates the rapid loss of Biradical X [N,N'-bis(1-oxyl - 2,2,5,5 - tetramethylpyrroline-3-carboxy)-1,2-diaminoethane] and Monradical A (2,2,5,5-tetramethyl-3-carbamidpyrroline-1-oxyl). However, the 5-line EPR spectrum characteristic of Biradical X in aqueous solution was changed to a dominantly 3-line spectrum within a few minutes after illumination in the presence of ferredoxin and NADP+. Analysis of the double integration of the first derivative EPR spectrum revealed no decrease in Biradical X concentration for more than 30 min of illumination. Our data suggest that Biradical X attaches to some soluble macromolecule(s) and that illumination of chloroplasts promotes such an attachment.     

Photosynthetic electron transport from water to NADP driven by photosystem II in inside-out chloroplast vesicles

It is now widely held that the light-induced noncyclic (linear) electron transport from water to NADP⁺ requires the collaboration in series of the two photosystems that operate in oxygen-evolving cells: photosystem II (PSII) photooxidizes water and transfers electrons to photosystem I (PSI); PSI photoreduces ferredoxin, which in turn reduces NADP⁺ (the Z scheme). However, a recently described alternative scheme envisions that PSII drives the noncyclic electron transport from water to ferredoxin and NADP⁺ without the collaboration of PSI, whose role is limited to cyclic electron transport [Arnon, D. I., Tsujimoto, H. Y. & Tang, G. M.-S. (1981) Proc. Natl. Acad. Sci. USA 78, 2942-2946]. Reported here are findings at variance with the Z scheme and consistent with the alternative scheme. Thylakoid membrane vesicles were isolated from spinach chloroplasts by the two-phase aqueous polymer partition method. Vesicles, originating mainly from appressed chloroplast membranes that are greatly enriched in PSII, were turned inside-out with respect to the original sidedness of the membrane. With added plastocyanin, ferredoxin, and ferredoxin-NADP⁺ reductase, the inside-out vesicles enriched in PSII gave a significant photoreduction of NADP⁺ with water as electron donor, under experimental conditions that appear to exclude the participation of PSI.     

Distinct features of dehydrocorticosterone reduction into corticosterone in the liver and duodenum of the domestic fowl (Gallus gallus domesticus)

The mammalian 11-beta hydroxysteroid dehydrogenase type 1 (11 betaHSD1) reduces glucocorticoids (GC) at C11 from the 11-keto-GC nonactive form to the 11-hydroxy-GC active form, an action essential for survival. Whereas GC metabolism at C11 and the role of 11 betaHSD1 are studied extensively in mammals, information about these in birds is scattered. Herein, we report the GC bidirectional metabolism in chickens. In hens' liver and duodenal mucosa, 11 betaHSD1-like mRNA expression was detected; and 11 betaHSD1-like immunoreactivity was found linked to membranes of hepatocytes and duodenal enterocytes. With either NADH or NADPH, the membranal fraction of liver and duodenal mucosa converted dehydrocorticosterone (A) into corticosterone (B) with K(m) (1.1-8.7 microM) and V(max) (10-40 pmol/mg protein/min) values similar to those reported for mammalian 11 betaHSD1. In the presence of NADP(+) or NAD(+), these membranal fractions oxidized B into A. With either NADPH or NADH, the cytosol of chicken liver and duodenal mucosa reduced A into B (K(m) of 1.1 - 2.3 microM and V(max) of 260-960 pmol/mg protein/min). These cytosolic fractions did not convert any amount of B into A when incubated with either NADP(+) or NAD(+). This may suggest that chicken liver and duodenal mucosa express 11 betaHSD1 that is a membrane-bound oxoreductase which uses both NADPH/NADP(+) and NADH/NAD(+) as cosubstrates. The substantial reduction of A into B (but no conversion of B into A) found in the cytosol is most likely executed by a unidirectional soluble reductase, different than 11 betaHSD1.     

Photoreduction of NADP+ Sensitized by Synthetic Pigment Systems

Two synthetic pigment systems capable of enzymatically photoreducing NADP(+) are described. One system contains proflavine; the other, acridine. The complete system consists of ethylene diamine tetraacetic acid (EDTA), proflavine (or acridine), ferredoxin-NADP reductase (EC 1.6.99.4), and NADP(+). The two pigments initiate the photoreduction of NADP(+) in different portions of the electromagnetic spectrum. Proflavine photosensitizes in the visible portion; acridine, in the ultraviolet. Neither proflavine nor acridine is structurally related to chlorophyll. The acridine system has the attractive property that the enzyme, ferredoxin-NADP reductase, is the only component of the system that absorbs appreciably in the visible region of the spectrum.     

On the iron-sulfur cluster in hydrogenase from Clostridium pasteurianum W5.

Hydrogenase, purified to an average specific activity of 328 mumol of H2 evolved/(min X mg of protein) from Clostridium pasteurianum W5, was found to have 4-5 Fe and 4-5 labile sulfur atoms per molecule of 60,000 molecular weight, in contrast with earlier reports of 12 Fe per molecule. Displacement of the iron-sulfur cluster from hydrogenase by thiophenol in 80% hexamethyl phosphoramide:20% H2O yielded the Fe4S4 (thiophenyl)4 dianion according to absorption spectroscopy. Electron paramagnetic resonance spectroscopy at 12 K showed that the iron-sulfur cluster in the enzyme could be reduced by the H2 to a state (g-values of 2.098, 1.970, and 1.898) similar to that in reduced ferredoxin and could be oxidized by dichlorophenolindophenol or H+ to a state (g-values at 2.099, 2.041, and 2.001) similar to that in high potential iron-sulfur proteins. These oxidations and reductions appeared to occur within the turnover time of the enzyme. Deuterium failed to narrow the electron paramagnetic resonance signal in either state, but the competitive inhibitor carbon monoxide reversibly formed a compound with either state and substantially altered the electron paramagnetic resonance. 13CO produced a broadening of these signals, suggesting the formation of a direct CO complex with the iron-sulfur cluster. These data are consistent with a model of the active site of the enzyme in which a four-iron four-sulfur cluster is a component that can accept one or two electrons from and donate either one or two electrons to substrates, and in which the iron-sulfur cluster serves as the site of binding of gaseous ligands.     

The Isolation and Characterization of a New Iron-Sulfur Protein from Photosynthetic Membranes

A new iron-sulfur protein, distinct from the soluble chloroplast ferredoxin, was isolated from chloroplast membranes. The isolated protein, purified to homogeneity, had a molecular weight of about 8000 and 4 atoms of iron and 4 inorganic sulfides per mole. Its absorption spectrum had a broad absorbance band in the 400 nm region, a shoulder at approximately 310 nm, and a peak around 280 nm. The absorbance ratio A₄₀₀ to A₂₈₀ was 0.55. The electron paramagnetic resonance spectrum (measured at 12°K) of the reduced protein was similar to that of other reduced iron-sulfur proteins, showing a major resonance line at g = 1.94.     

Cytochrome b-559 and proton conductance in oxygenic photosynthesis

Although cytochrome b-559 has long been known as a membrane-bound redox component closely linked to the reaction center of the oxygen-generating photosystem (PSII), its role in photosynthesis has remained obscure. This paper reports evidence and outlines a hypothesis in support of a “b-559 cycle”—i.e., a light-induced, cytochrome b-559-dependent, cyclic electron transport pathway around PSII that promotes translocation of protons from plastoquinol into the aqueous domain (lumen) of photosynthetic membranes (thylakoids). Light-induced proton transport coupled to light-induced electron transport is an essential aspect of energy transduction in photosynthesis because it generates an electrochemical proton gradient that drives ATP synthesis by the process of photosynthetic phosphorylation. The principal carrier of electrons and protons in thylakoids is the plastoquinone/plastoquinol couple. We propose that the b-559 cycle functions as a redox-linked proton pump that may operate jointly with the Rieske iron-sulfur pathway in oxidizing plastoquinol. The overall effect of such concerted oxidation of plastoquinol would be the translocation into the thylakoid lumen of two protons for each electron transferred from water to plastocyanin via plastoquinone.     

Synthesis and Properties of Clostridium acidi-urici [Leu2]-Ferredoxin: A Function of the Peptide Chain and Evidence Against the Direct Role of the Aromatic Residues in Electron Transfer

Tyrosyl or other aromatic residues generally occur in two conserved positions in the peptide chain of clostridial-type ferredoxins and have been implicated in the electron transfer function of these iron-sulfur proteins. We have prepared and determined some of the properties of a derivative of Clostridium acidi-urici ferredoxin, [Leu(2)]-ferredoxin, in which a leucyl residue has been substituted for the tyrosyl residue in position 2 from the amino terminus. [Leu(2)]-ferredoxin is fully active as an electron carrier in two biological assays, the phosphoroclastic enzyme system and the ferredoxin-dependent reduction of cytochrome c in the presence of ferredoxin-TPN reductase and TPNH. Quantitative electron paramagnetic resonance experiments indicate that [Leu(2)]-ferredoxin accepts nearly two electrons upon enzymatic reduction by pyruvate-ferredoxin oxidoreductase and an excess of pyruvate. If electron transfer to an iron-sulfur cluster is the rate-limiting step in the assays used, and if the rate of electron transfer through Tyr(30) is not much faster than through Tyr(2), these results indicate that the primary pathway of electron transfer in clostridial-type ferredoxins is not via Tyr or other aromatic amino-acid residues. The syntheses of other ferredoxin derivatives with amino-acid substitutions or deletions in positions 1 and 2 indicate that a large bulky residue, but not necessarily an aromatic residue, is needed in position 2 for the stability of this ferredoxin. The residue in position 2, therefore, appears to act as a hydrophobic shield for an iron-sulfur cluster.     

Formation of the iron-sulfur cluster of ferredoxin in isolated chloroplasts

The formation of the iron-sulfur cluster of ferredoxin was examined in vitro by incubating isolated chloroplasts with [³⁵S]cysteine. The ferredoxin molecule was radioactively labeled in chloroplasts without synthesis of its polypeptide and comigrated with holoferredoxin during polyacrylamide gel electrophoresis under nondenaturing conditions. When the labeled ferredoxin was denatured by the addition of trichloroacetic acid, radioactive acid-labile sulfide in the cluster was released from the polypeptide as a gas and trapped in a 0.1 M NaOH solution. These results indicate that the sulfur atom derived from cysteine was incorporated into ferredoxin through formation of the iron-sulfur cluster. This process was stimulated by light and inhibited by the electron transport inhibitor, dichlorophenyldimethylurea, and the uncouplers, atebrin and gramicidin, but not by the protein synthesis inhibitor, chloramphenicol. These inhibitory effects were reversed by the addition of ATP to the incubation mixture. Formation of the iron-sulfur cluster of ferredoxin in chloroplasts is thus dependent on ATP.     

Regulation of ferredoxin-catalyzed photosynthetic phosphorylations.

Under aerobic conditions that are likely to prevail in chloroplasts in vivo, the optimal concentration of ferredoxin for cyclic photophosphorylation was found to be equal to that required for NADP reduction and about one-tenth of that needed for cyclic photophosphorylation under anaerobic conditions. In the presence of ferredoxin and NADP, cyclic photophosphorylation operated concurrently with noncyclic photophosphorylation, producing an ATP: NADPH ratio of about 1.5. The effective operation of ferredoxin-catalyzed cyclic photophosphorylation by itself required a curtailment of the electron flow from water which was accomplished experimentally by the use of either an inhibitor or far-red monochromatic light. An unexpected discovery was that the operation of cyclic photophosphorylation by itself was also regulated by a back reaction of NADPH and ferredoxin with two components of chloroplast membranes, component C550 and cytochrome b559. The significance of these findings to photosynthesis in vivo is discussed.