Low-potential Reductants Research Articles

Can Photosystem II be a photogenerator of low potential reductant for CO2 fixation and H2 photoevolution?

The experimental grounds of the hypothesis of a single-light-reaction in oxygenic photosynthesis as suggested by Greenbaum et al. (Nature (1995) 376: 438-441) are critically discussed and a possible explanation of their data by Photosystem I contamination in the mutant utilized is argued.

An improved method for fluoro-dediazoniation of arylamines substituted with polar groups

The addition of SnX 2 (SnCl 2 or SnF 2), as a low redox potential reductant, at the fluoro-dediazoniation step in the deaminative fluorination of aryl amines substituted with polar groups gave high selectivities for the formation of fluoroaromatics under mild conditions. The selectivities were further increased by the addition of a nucleophilic fluoride anion source, i.e. tetrabutylammonium dihydrogen trifluoride nBu 4N + H 2F 3 −, along with the reductants.

Characterization of the CO oxidation/H2 evolution system of Rhodospirillum rubrum. Role of a 22-kDa iron-sulfur protein in mediating electron transfer between carbon monoxide dehydrogenase and hydrogenase.

The response of the membrane-associated carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum to solubilization by detergents and organic solvents, the properties of solubilized CODH, and the mechanism for coupling CO oxidation to hydrogen evolution via a CO-induced hydrogenase activity have been investigated. The release of CODH by a variety of ionic and nonionic detergents occurs in a redox-dependent fashion: CODH is solubilized in the presence of low-potential reductants (dithionite, CO, and H2) but is resistant to solubilization from membranes prepared in the absence of reductant or membranes prepared in the presence of reductant and subsequently dye-oxidized. This redox-dependent response to detergent solubilization has been exploited to release CODH from the membranes in a purified state. CODH can also be solubilized from deoxycholate-washed membranes in a redox-independent manner with 20% ethanol. CODH solubilized by deoxycholate or ethanol, when purified to homogeneity by the protocol previously described for heat-solubilized CODH (Bonam, D., and Ludden, P. W. (1987) J. Biol. Chem. 262, 2980-2987), is associated with a previously unobserved 22-kDa protein. The 22-kDa protein can be dissociated from CODH with acetonitrile and can be reconstituted with CODH, after removal of acetonitrile, in a stoichiometric (1:1) fashion. The isolated 22-kDa protein contained 4.0 iron atoms, a reducible Fe-S center, and was O2- and heat-labile. The 22-kDa protein did not alter the catalytic properties of CODH as assayed in vitro with methyl viologen as the electron acceptor for CO oxidation, but was required for reconstituting CO oxidation to hydrogen evolution via the CO-induced membrane-bound hydrogenase. Other electron carrier proteins (ferredoxins and flavodoxin) were ineffective at coupling CO oxidation and hydrogen evolution. We conclude that the 22-kDa protein is a reversibly dissociable subunit of CODH tha mediates electron transfer to hydrogenase.

Absorption spectra of radical forms of 2,4-dihydroxybenzoic acid, a substrate for p-hydroxybenzoate hydroxylase

Combined optical and conductimetric measurements in aqueous solution indicate that at high pH (greater than or equal to 10).OH radicals react with the phenoxide form of 2,4-dihydroxybenzoic acid to form transiently phenoxyl radicals and a small amount of hydroxyeyclohexadienyl (HCHD) radicals by 150 ns. The respective yields of 88 and 12% of the total.OH radical yield were deduced from conductance and optical changes as well as from studies using a low potential reductant. The HCHD radical possesses a pKa of 8.0 +/- 0.1 and the constructed spectrum of the deprotonated forms of HCHD has a lambda max at 420 nm with a minimum extinction coefficient of approximately 7250 M-1 cm-1. The red shift in lambda max and increase in extinction coefficient compared to the revised spectral properties of the protonated form of the HCHD radical (lambda max at 390 nm with extinction coefficient of approximately 4500 M-1 cm-1), together with the pKa of the HCHD radical, provide an explanation for the pH-dependent spectral changes of the so-called highly absorbing intermediate II species, observed in the functioning of the enzyme p-hydroxybenzoate hydroxylase. These results add further to the evidence in support of the proposal that intermediate II is composed of species which absorb similarly to the flavin 4(a)-hydroxide and a form of the substrate/product such as the HCHD radical (Anderson, R. F., Patel, K. B., and Stratford, M. R. L. (1987) J. Biol. Chem. 262, 17475-17479).

Redox reactivity of bacterial and mammalian ferritin: is reductant entry into the ferritin interior a necessary step for iron release?

Both mammalian and bacterial ferritin undergo rapid reaction with small-molecule reductants, in the absence of Fe2+ chelators, to form ferritins with reduced (Fe2+) mineral cores. Large, low-potential reductants (flavoproteins and ferredoxins) similarly react anaerobically with both ferritin types to quantitatively produce Fe2+ in the ferritin cores. The oxidation of Fe2+ ferritin by large protein oxidants [cytochrome c and Cu(II) proteins] also occurs readily, yielding reduced heme and Cu(I) proteins and ferritins with Fe3+ in their cores. These latter oxidants also convert enthetically added Fe2+, bound in mammalian or bacterial apo- or holoferritin, to the corresponding Fe3+ state in the core of each ferritin type. Because the protein reductants and oxidants are much larger than the channels leading into the mineral core attached to the ferritin interior, we conclude that redox reactions involving the Fe2+/Fe3+ components of the ferritin core can occur without direct interaction of the redox reagent at the mineral core surface. Our results also suggest that the oxo, hydroxy species of the core, composed essentially of Fe(O)OH, arise exclusively from solvent deprotonation. The long-distance ferritin-protein electron transfer observed in this study may occur by electron tunneling.

H + transport in the evolution of photosynthesis

Extant photosynthetic organisms all appear to use transmembrane H + fluxes as the coupling agent in the use of light energy in ATP synthesis. In the steady-state there is a large H + free energy difference across the coupling membrane, and when this is reflected as a light-induced change in pH of the phase (cytosol or stroma) containing the enzymes of carbon assimilation, the H + transport can have an informational role in activating and inactivating enzymes. The earliest organisms probably lived fermentatively (substrate-level phosphorylation) in an anaerobic environment provided with organic solutes synthesised abiotically. There are good reasons for believing that one of the earliest primary active transport systems (interconverting chemical and electrical/osmotic energy) was an H + extrusion pump powered by ATP or PP i. Its initial function was extrusion of excess H + from the fermenting cells, and the support of a number of co-transport processes. The earliest energetic use of light energy is envisaged as being the energization of an alternative H + extrusion pump, with bacteriorhodopsin or (bacterio-) chlorophyll as the pigment. The former type of cyclic photoredox system ( Halobacterium-type) is simpler than the latter: a “pre-respiratory” chemical redox H + pump may have preceded the (bacterio-) chlorophyll-based process. Any of these H + pumps could spare the use of fermentative ATP in powering active H + efflux and would thus have been favoured as fermentative substrates became scarce; eventually the larger Δ μ H + generated by the light-powered H + pump was used to drive the ATP-powered H + pump backwards and thus generate ATP with light as the ultimate energy source. Scarcity of suitable reductants for biosynthesis as life proliferated provided a selective impetus for a non-cyclic photoredox system which could use light energy to generate a low-potential reductant at the expense of more readily available higher-potential reductants. The non-cyclic photoredox system is not possible in its simplest form (with all the redox energy coming from excitation energy of one or more photoreactions) in the bacteriorhodopsin line of evolution. Such a simple photoredox system is found in the Chlorobiaceae; even if (as seems likely) the non-cyclic photoredox process generates a Δ μ H + (and thus, potentially, ATP), some of the ATP needed for CO 2 fixation and cell growth must be generated by a cyclic photoredox system. In the extant purple bacteria the generation of low-potential reductant involves a non-cyclic photoredox pathway which produces a reductant unable to reduce NAD +; the “energy gap” is spanned by “reverse electron transfer” which uses energy from a Δ μ H + . It is not clear if this energetic requirement for the H + gradient can be quantitatively satisfied from a non-cyclic photoredox H + transport; it is certain that there is a major requirement for cyclic photoredox H + pumping in these organisms. The photosynthetic bacteria are today restricted to reducing (low E h ) environments similar to those found in the early, anoxic earth; they are unable to use very weak reductants as donors for non-cyclic photoredox processes. As the sources of even weakly reducing donors (other than H 2O) on the primitive earth were depleted the two photoreactions scheme of extant O 2-producers evolved by modification of the bacterial photoreaction. This non-cyclic photoredox process is definitely H +-translocating and the role of cyclic photoredox processes in ATP generation in O 2-evolvers is smaller than in photosynthetic bacteria. In parallel with the biochemical and biophysical changes in the photosystems there was a morphological evolution, with an increasing tendency for “internalisation” of the photoredox processes (originally present in the plasma membrane, as in extant Chlorobineae) into thylakoids (as in most Rhodospirillineae, Cyanobacteria and in all eukaryotes). With a plasmalemma-located photoredox system, and the constraints of a fixed, alkaline external pH and the cytoplasmic pH of 7–8, the Δ μ H + would be generated largely as an electrical P.D. The presence of a phase (intrathylakoid space) with a “negotiable pH” would permit the generation and use of a Δ μ H + largely present as a pH gradient. In both cases illumination can cause an increase in cytoplasmic (stromal) pH over the dark value; this is an important aspect of the regulation of “phototrophic” and “heterotrophic” enzyme systems in the light and in the dark. However, it is argued that these differences in pH are not absolutely light-dependent unless they depend upon some more uniquely light-dependent signal, probably based on a redox component only generated in the light.

The thermochemical characterization of sodium dithionite, flavin mononucleotide, flavin-adenine dinucleotide and methyl and benzyl viologens as low-potential reductants for biological systems.

The heat of reaction (deltaH) of Fe(CN)63-, Methyl Viologen, FMN and FAD with S2O42- in aqueous buffer solutions was measured calorimetrically. In addition deltaH values for reduction of Fe(CN)63-, FMN and FAD by reduced Methyl Viologen were determined. The resulting calorimetric data and corresponding E0 values were combined to yield thermodynamic data for these simple reducing agents in a form useful for applications to biological reactions. Thermodynamic data for the reduction of spinach ferredoxin are also presented.

Primary electron acceptor complex of photosystem I in spinach chloroplasts

PHOTOSYSTEM I (PSI) is the photochemical reaction complex involved in the generation of a low potential reductant in oxygen-evolving photosynthetic organisms. The primary photochemical reaction in PSI has been identified as the photo-oxidation of a reaction centre chlorophyll complex P700 (ref. 1). This photo-oxidation occurs both at room temperature and in the frozen state at temperatures as low as 4.2 K. At cryogenic temperatures this photo-oxidation has generally been found to be irreversible2. Malkin and Bearden3 showed that the electron acceptor for this photo-oxidation reaction is a bound ferredoxin. This ferredoxin has two iron–sulphur centres with very low redox potentials (Em10 = −550 and −590 mV)4–6. It has been proposed that these centres are the primary electron acceptor of PSI3,4. More recent evidence suggests it may not be7–11, and we have shown11 that when the bound ferredoxin is chemically reduced, illumination at low temperature results in the photo-oxidation of P700 without any change in the bound ferredoxin, this oxidation is reversible. In photosynthetic bacteria a component with an EPR signal at g = 1.82 has been identified as the primary electron acceptor of the photochemical system12,13 and it is possible that a similar component might be involved in chloroplast photochemical reactions. McIntosh et al.14 obtained kinetic evidence for the presence in PSI particles of a component with an EPR signal at g = 2.06 and g = 1.76 which showed a reversible redox change on illumination of the particles at low temperature parallel to that of P700. The changes observed were small and it was not possible to obtain a spectrum of the component. Using a more highly purified PSI preparation we have now obtained strong evidence that this component is the primary electron acceptor of PSI.

Photosynthetic Production of Hydrogen Peroxide by Anacystis nidulans

A sensitive assay based upon fluorescence of scopoletin allowed continuous recording of H(2)O(2) production in illuminated intact cells of Anacytis nidulans. Onset of illumination was followed by a 5 to 10 second lag, a burst of very rapid production continuing for up to 5 minutes, and finally a slow and continuing steady rate of H(2)O(2) production. Size of the H(2)O(2) burst was decreased by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, by low O(2), and by certain Calvin cycle intermediates; it was increased by high light intensity, CO(2) depletion, Calvin cycle inhibitors (as iodoacetamide), cold shock, carbonyl cyanide m-chlorophenylhydrazone, and certain organic acids as glycolate). The H(2)O(2) burst was explained by the following hypothesis: a low potential reductant is produced more rapidly than it can be used in the normal pathway to CO(2) reduction and, instead, reacts with oxygen. H(2)O(2) production is regarded as a metabolic defect observable in Anacystis most dramatically during the transition from a very low rate of oxidative dark metabolism to a high rate of photosynthetic metabolism.

Nitrogenase activity in cell-free extracts of the blue-green alga, Anabaena cylindrica.

Cell-free extracts with high nitrogenase activity were prepared by sonic oscillation and French press treatment from the blue-gree alga Anabaena cylindrica. Extracts were prepared from cells grown on a 95% N(2)-5% CO(2) gas mixture followed by a period of nitrogen starvation under an atmosphere of 95% argon-5% CO(2). No increase in the specific activity of extracts was achieved by breaking heterocysts. Activity (assayed by acetylene reduction) was found to be dependent on adenosine triphosphate (ATP), an ATP-generating system, and a low-potential reductant. Na(2)S(2)O(2) employed as reductant supports higher rates of nitrogenase activity than reduced ferredoxin. The activity is associated with a small-particle fraction that can be sedimented by ultracentrifugation. In contrast to the particulate nitrogenase of Azotobacter, which is stable in air, the A. cylindrica nitrogenase is an oxygen sensitive as nitrogenase prepared from anaerobic bacteria.