Energetics of the one-electron steps in the NAD+/NADH redox couple

The one-electron reduction potential of NAD⁺ has been determined using pulse radiolysis to study electron-transfer equilibria between it and a low potential bipyridylium compound. The determined value E¹₇ (NAD⁺/NAD^.) = ?922 ± 8 mV (NHE scale) is used to calculate E²₇ (NAD^./NADH) = +282 mV. E¹₇ for 1-methylnicotinamide, E¹₇ (MeN⁺/MeN^.) = ?918 ± 7 mV.     

Voltage dependent potassium fluxes and the significance of action potentials in Acetabularia

Membrane potential, V_m, and K⁺(⁸⁶Rb⁺) fluxes have been measured simultaneously on individual cells of Acetabularia mediterranea. During resting state (resting potential approx. ?170 mV) the K⁺ influx amounts to 0.24–0.6 pmol · cm^?2 · s^?1 and the K⁺ efflux to 0.2–1.5 pmol · cm^?2 s^?1. According to the K⁺ concentrations inside and outside the cell (40 : 1) the voltage dependent K⁺ flux (zero at V_m = E_K = ?90 mV) is stimulated approx. 40-fold for V_m more positive than E_K.It is calculated that during one action potential (temporary depolarization to V_m more positive than E_K) a cell looses the same amount of K⁺, which leaks in during 10–20 min in the resting state (V_m = ?170 mV). Since action potentials occur spontaneously in Acetabularia, they are therefore suggested to have a significant function for the K⁺ balance of this alga.     

The one-electron reduction potentials of NAD

The one-electron reduction potential (E¹₇) of NAD⁺ has been determined by pulse radiolysis to be ?0.94 V. E²₇ (E¹₇ for the free radical, NAD^.) is +0.30 V. E¹₇ for 1-methylisonicotinamide is ?0.77 V.     

Pulse radiolysis studies of antitumor quinones: Radical lifetimes,reactivity with oxygen,and one-electron reduction potentials

The formation of semiquinone free radicals from antitumor drugs has been studied by pulse radiolysis. The semiquinone free radicals are reactive and have short half-lives in aqueous media under anaerobic conditions. The half-lives of the radicals formed from adriamycin, mitomycin C, and 2,5-diaziridinyl-3,6-bis(carboethoxy)amine-1,4-benzoquinone (AZQ) are 50,100, and 200 μs, respectively. The mean diffusion distance of the semiquinone free radical is less than 0.6 μm. In the presence of molecular oxygen the half-life of the semiquinone free radical is shortened. Adriamycin semiquinone reacts rapidly with oxygen, k = 4.4 × 10⁷m^?1s^?1. In air-saturated buffer the half-life of adriamycin semiquinone radical can be calculated to be 8 μs with a mean diffusion distance of less than 0.1 μm. If the half-lives in buffer are comparable to those within a cell, semiquinone free radicals must be generated close to the site at which they produce a biological effect. One-electron reduction potentials (E₇¹) were determined and were AZQ, ?168 mV, adrenochrome, ?253 mV, mitomycin C, ?271 mV, adriamycin, ?292 mV, daunomycin, ?305 mV, and anthracenedione, ?348 mV. Enzymatic one-electron reduction of these antitumor quinones by NADPH-cytochrome P-450 reductase increased at more positive values of quinone E₇¹.     

One-electron reduction of adriamycin: Properties of the semiquinone

Pulse radiolysis of aqueous solutions containing adriamycin and redox indicators of known one-electron reduction potential (E¹) shows that its E¹ at pH 7 is ?328 mV (vs NHE). The variation E¹ with pH in the range 6–12 shows that the net charge on the semiquinone at pH 7 is zero. As well as the pK_a values of 2.9 and ≥ 14 established independently, the semiquinone has a pK_a close to 9.2. The new data enable the structure and likely reactivity of the semiquinone to be specified.     

Function of two β-carotenes near the D1 and D2 proteins in photosystem II dimers

The antenna proteins in photosystem II (PSII) not only promote energy transfer to the photosynthetic reaction center (RC) but provide also an efficient cation sink to re-reduce chlorophyll a if the electron transfer (ET) from the Mn-cluster is inhibited. Using the newest PSII dimer crystal structure (3.0 Å resolution), in which 11 β-carotene molecules (Car) and 14 lipids are visible in the PSII monomer, we calculated the redox potentials (E_m) of one-electron oxidation for all Car (E_m(Car)) by solving the Poisson-Boltzmann equation. In each PSII monomer, the D1 protein harbors a previously unlocated Car (Car_D1) in van der Waals contact with the chlorin ring of Chl_Z(D1). Each Car_D1 in the PSII dimer complex is located in the interface between the D1 and CP47 subunits, together with another four Car of the other PSII monomer and several lipid molecules. The proximity of Car bridging between Car_D1 and plastoquinone/Q_A may imply a direct charge recombination of Car⁺Q_A⁻. The calculated E_m(Car_D1) and E_m(Chl_Z(D1)) are, respectively, 83 and 126 mV higher than E_m(Car_D2) and E_m(Chl_Z(D2)), which could explain why Car_D2⁺ and Chl_Z(D2)⁺ are observed rather than the corresponding Car_D1⁺ and Chl_Z(D1)⁺.     

A study of the current-voltage relationships of electrogenetic active and passive membrane elements in riccia fluitans

Potassium- and proton-dependent membrane potential, conductance, and current-voltage characteristics (I–V curves) have been measured on rhizoid cells of the liverwort Riccia fluitans. The potential difference (E_m) measured with microelectrodes across plasmalemma and tonoplast is depolarized to the potassium-sensitive diffusion potential (E_D) in the presence of 1 mM NaCN, 1 mM NaN₃, or at temperatures below 6°C. Whereas the temperature change from 25°C to 5°C decreases the membrane conductance (g_m) from 0.71 to 0.43 S ? m^?2, 1 mM NaCN increases g_m by about 25%. The membrane displays potassium-controlled rectification which gradually disappears at temperatures below 5°C. The potassium pathway can be described by an equivalent circuit of a diode and an ohmic resistor in parallel. In the potential interval of E_D ± 100 mV the measured I-V curves roughly fit the theoretical curves obtained from a modified diode equation. ⁸⁶Rb⁺(K⁺)-influx is voltage sensitive: In the presence of 1 mM NaCN, ⁸⁶Rb⁺-influx follows a hyperbolic function corresponding to a low conductance at low [K⁺]_o and high conductance at high [K⁺]_o. On the contrary ⁸⁶Rb⁺-influx is linear with [K⁺]_o when pump activity is normal. It is believed that there are two K⁺-transport pathways in the Riccia membrane, one of which is assigned to the low conductance (0.2 S · m^?2), the other to a temperature-dependent facilitated diffusion system with a higher conductance (7.7 S · m^?2). The electrogenic pump essentially acts as a current source and consumes about 39% of the cellular ATP-turnover. In the presence of 30 μM CCCP the saturation current of 0.1 A · m^?2 is doubled to about 0.2 A · m^?2, and the electromotive force of ?360 mV switches to ?250 mV. It is suggested that this may be due to a change in stoichiometry from one to two transported charges per ATP hydrolyzed.     

Oxidation-reduction properties of the electron acceptors of Photosystem II. II. Redox titration at various pH values of the flash-induced formation of C550 in Chlamydomonas Photosystem II particles lacking the secondary quinone electron acceptor

Redox titrations of the flash-induced formation of C550 (a linear indicator of Q^?) were performed between pH 5.9 and 8.3 in Chlamydomonas Photosystem II particles lacking the secondary electron acceptor, B. One-third of the reaction centers show a pH-dependent midpoint potential (E_m,7.5) = ? 30 mV) for redox couple $\text{Q}\text{Q}{}^{\mathrm{?}}$, which varies by ?60 mV/pH unit. Two-thirds of the centers show a pH-independent midpoint potential (E_mm = + 10 mV) for this couple. The elevated pH-independent E_m suggests that in the latter centers the environment of Q has been modified such as to stabilize the semiquinone anion, Q^?. The midpoint potentials of the centers having a pH-dependent E_m are within 20 mV of those observed in chloroplasts having a secondary electron acceptor. It appears therefore that the secondary electron acceptor exerts little influence on the E_m of $\text{Q}\text{Q}{}^{\mathrm{?}}$. An EPR signal at g 1.82 has recently been attributed to a semiquinone-iron complex which comprises Q^?. The similar redox behavior reported here for C550 and reported by others (Evans, M.C.W., Nugent, J.H.A., Tilling, L.A. and Atkinson, Y.E. (1982) FEBS Lett. 145, 176–178) for the g 1.82 signal in similar Photosystem II particles confirm the assignment of this EPR signal to Q^?. At below ?200 mV, illumination of the Photosystem II particles produces an accumulation of reduced pheophytin (Ph^?). At ?420 mV Ph^? appears with a quantum yield of 0.006–0.01 which in this material implies a lifetime of 30–100 ns for the radical pair P-680⁺Ph^?.     

The effect of redox potential of the kinetics of fluorescence induction in pea chloroplasts I. Removal of the slow phase

The effect of alteration of redox potential on the kinetics of fluorescence induction in pea chloroplasts has been investigated. Potentiometric titration of the initial (F_i) level of fluorescence recorded upon shutter opening gave a two component curve, with E_m(7) at ?20 mV and ?275 mV, almost, identical to results obtained using continuous low intensity illumination (Horton, P. and Croze, E. (1979) Biochim. Biophys. Acta 545, 188–201). The slow or tail phase of induction observed in the presence of DCMU can be eliminated by poising the redox potential at approx. 0 to +50 mV. At this potential F_i was increased by less than 10% and the higher potential quencher described above was only marginally reduced. The disappearance of the slow phase titrated as an n = 1 component with an E_m(7) of +120 mV. Therefore it seems unlikely that the slow phase of fluorescence induction is due to photoreduction of the ?20 mV quencher. These results are discussed with reference to current ideas concerning heterogeneity on the acceptor side of Photosystem II.     

Studies of the function of the membrane-bound iron-sulfur centers of the photosynthetic bacterium Chromatium vinosum

Chromatophores from the photosynthetic bacterium, Chromatium vinosum, have been prepared which photoreduce NAD⁺ with either succinate or reduced dichlorophenolindophenol as electron donors. NAD⁺ reduction is inhibited by uncouplers as well as inhibitors of cyclic photophosphorylation. These chromatophores contain several bound iron-sulfur centers which have been detected by low-temperature EPR spectroscopy. One center, having a g 2.01 EPR signal in the oxidized state, has E_m7.5 = +50 mV and is partially reduced by succinate in the dark. Three iron-sulfur centers having g 1.93 EPR signals have been resolved by redox titration, and the E_m7.5 values of these centers are ?50, ?175 and ?250 mV, respectively. Studies of the involvement of these centers in electron transfer from donors to NAD⁺ have indicated that the center with E_m = ?50 mV is succinate reducible in the dark and appears to be analogous to center S-1 of succinic dehydrogenase in other systems. An additional g 1.93 iron-sulfur center can be photoreduced in the presence of electron donors and this reduction is inhibited by uncouplers. The possible role of the two low-potential iron-sulfur centers in relation to the dehydrogenases functioning in NAD⁺ reduction is considered.     

A new cytochrome b-like pigment with a peak at 567 nm and a low redox potential in denitrifying bacteria

Dithionite reduced difference spectra of extracts of denitrifying pseudomonads revealed small absorption maxima at 567 and 539 nm, suggestive of α and β bands of a new b type cytochrome. The new pigment was present in cells grown both aerobically and anaerobically and was located in the particulate fraction of extracts. These extracts also contained, in much higher concentrations, additional pigments resembling cytochromes c₅₅₃ and b₅₅₉, which were readily reduced by NADH or endogenous substrates, although a small proportion of the b₅₅₉ required dithionite for complete reduction. In contrast, most of the new 567 pigment was not readily reduced by NADH, succinate, or endogenous substrates, and it was most easily visualized with dithionite in the sample cuvette, and either endogenous substrates or NADH in the reference cuvette. Dyes of low redox potential such as benzyl viologen (E_m,7 = ?359 mV), phenosafranine (E_m,7 = ?250 mV) and reduced janus green (E_m,7 = ?225 mV) could substitute for dithionite as reductant for the new 567 pigment. Cresyl violet (E_m,7 = ?160 mV) caused partial reduction. However, redox compounds of higher potential such as reduced indigo carmine, (E_m,7 = ?125 mV) reduced methylene blue (E_m,7 = ?11 mV), ferrooxalate and ascorbate could not replace dithionite as reductant. Most of the cytochrome b₅₅₉ and the c₅₅₃ were reduced by ascorbate. Thus the new 567 pigment appears to have a mid-point potential between ?225 and ?125 mV, well below most of the cytochrome b₅₅₉. The new 567-nm pigment was rapidly oxidized by brief but vigorous aeration and was also slowly and partially re-reduced when concentrated extracts were allowed to stand without aeration. A more complete reduction of the 567 pigment was readily obtained by the addition of a mixture of NADH and FAD. The 567 pigment was observed in several denitrifying pseudomonads, P. fluorescens, P. stutzeri and also in Micrococcus denitrificans, but was not detectable in the non-denitrifiers Escherichia coli or Aerobacter aerogenes.     

A novel mode of lactate metabolism in strictly anaerobic bacteria

Lactate is a common substrate for major groups of strictly anaerobic bacteria, but the biochemistry and bioenergetics of lactate oxidation is obscure. The high redox potential of the pyruvate/lactate pair of E₀′ = ?190 mV excludes direct NAD⁺ reduction (E₀′ = ?320 mV). To identify the hitherto unknown electron acceptor, we have purified the lactate dehydrogenase (LDH) from the strictly anaerobic, acetogenic bacterium Acetobacterium woodii. The LDH forms a stable complex with an electron‐transferring flavoprotein (Etf) that exhibited NAD⁺ reduction only when reduced ferredoxin (Fd^2?) was present. Biochemical analyses revealed that the LDH/Etf complex of A. woodii uses flavin‐based electron confurcation to drive endergonic lactate oxidation with NAD⁺ as oxidant at the expense of simultaneous exergonic electron flow from reduced ferredoxin (E₀′ ≈ –500 mV) to NAD⁺ according to: lactate + Fd^2? + 2 NAD⁺ → pyruvate + Fd + 2 NADH. The reduced Fd^2? is regenerated from NADH by a sequence of events that involves conversion of chemical (ATP) to electrochemical and finally redox energy (Fd^2? from NADH) via reversed electron transport catalysed by the Rnf complex. Inspection of genomes revealed that this metabolic scenario for lactate oxidation may also apply to many other anaerobes.     

Effect of oxidation-reduction potential on light-induced cytochrome and bacteriochlorophyll reactions in chromatophores from the photosynthetic green bacterium Chlorobium

The reaction center bacteriochlorophyll of Chlorobium thiosulfatophilum has a midpoint oxidation-reduction potential (E_m) of +330 mV. Its photooxidation is unaffected by oxidation-reduction potentials in the range from +260 mV to ?70 mV but on further reduction is attenuated to zero in a one-electron transition with an E_m of ?130 mV.A c-type cytochrome with an E_m of +220 mV and absorption maxima at 551–552 nm (α-band) and 420 nm (γ-band) is present in Chlorobium chromatophores and undergoes photooxidation. Cytocrome c photooxidation is attenuated to zero in two 1-electron steps with E_m of +30 mV and ?130 mVPossible roles for +30 mV and ?130 mV components in photosynthetic electron transport in Chlorobium are discussed.     

Molybdenum doubly bridged and chelate nitrosyl complexes incorporating saturated n-alkanediolate ligands

The redox-active doubly bridged species [{Mo(NO)(Tp^Me2)Q}₂] [Tp^Me2 = tris(3,5-dimethylpyrazol-1-yl)hydroborate, Q = O(CH₂)_nO, n = 3, 5, or OCH₂(CF₂)_n−2CH₂O, n = 5, 6], and a chelate complex [Mo(NO)-(Tp^Me2)O(CH₂)₅O] were prepared and characterised by elemental and mass analyses, ¹H NMR and IR spectroscopy. The bimetallic species with C₃, C₅, and C₅(F) bridges exhibit two well-resolved reduction processes in their cyclic voltammograms (ΔE_1/2 values of 290, 170, and 170 mV, respectively). These results indicate that the presence of the second bridge increases the extent of electrochemical interactions (by ca. 90-130 mV) in comparison with their singly bridged analogues. All non-fluorinated and the chelate species were catalytically active in cathodic reduction of chloroform.     

Oxidation-reduction potentials and spectral properties of some cytochromes from Thiobacillus versutus (A2)

Cytochromes c-550 (acidic), c-550 (basic), c-551 and c-552.5 from Thiobacillus versutus have been highly purified and characterized. Their spectral properties at 77 K are described. Oxidation-reduction titrations of cytochromes c-550 (acidic) and c-550 (basic) showed them to exhibit Nernst values of n = 1, with single redox centres in the cytochromes, and to have midpoint redox potentials at pH 7.0 (E_m,7) of 290 and 260 mV, respectively. Cytochrome c-551 contained two separately titratable redox components, each giving n = 1. The low potential centre (55% of titratable cytochrome) and the high potential centre (45%) had E_m,7 values of ?115 and +240 mV, espectively. Cytochrome c-552.5 also contained at least two redox centres. One (65% of titratable cytochrome) had n = 1 and E_m,7 = 220mV. The remaining 35% appeared to be a low potential component with an E_m,7 possibly as low as ?215 mV. the roles of these cytochromes in respiratory thiosulphate oxidation are discussed.     

Electrochemical reduction of rhenium(V) dithiocarbamate complexes trans-Re2O3(S2CNR2)4 in nonaqueous media

The electrochemistry for the reduction of tetrakis(dialkyl- and diphenyldithiocarbamato)-μ-oxodioxodirhenium complexes trans-Re₂O₃(S₂CNR₂)₄ (R = methyl, ethyl, propyl, butyl and phenyl) was investigated in seven nonaqueous solvents. The complexes underwent a reversible reduction involving one- electron at a platinum electrode to [Re₂O₃(S₂CNR₂)₄]⁻, which decomposed with the cleavage of the μ-oxo bridge to form ReO(S₂CNR₂)₂, R₂CNS₂⁻ and other rhenium complexes. The redox potential E^o′ of [Re₂O₃(S₂CNR₂)₄]^0/− couples and the stability of the reduction product [Re₂O₃(S₂CNR₂)₄]⁻ depend on the R group. The E^o′ are appreciably solvent-dependent. The difference in E^o′ with solvents could be interpreted in terms of the solubility parameters.     

Characterization of cytochrome b in the isolated ubiquinol-cytochrome c2 oxidoreductase from Rhodopseudomonas sphaeroides GA

Extinction coefficients for cytochrome b and c₁ in the isolated cytochrome bc₁ complex from Rhodopseudomonas sphaeroides GA have been determined. They are 25 mM^?1.cm^?1 at 561 nm for cytochrome b and 17.4 mM^?1.cm^?1 at 553 nM for cytochrome c₁ for the difference between the reduced and the oxidized state. Cytochrome b is present in two forms in the complex. One form has an E_m7 of 50 mV, an α-peak of 557 nm at liquid N₂ temperature and of 561 nm at RT, which is red-shifted by antimycin A. The other form has an E_m7 of ?90 mV, a double α-peak of 555 and 561 nm at liquid N₂ temperature corresponding to 559 and 566 nm at RT. The absorption at 566 nm is red-shifted by myxothiazol. The two shifts are independent of each other. Both midpoint potentials of cytochromes b are pH-dependent. The redox center compositions of the cytochrome bc₁ complexes from Rhodopseudomonas sphaeroides and from mitochondria are identical.