An adaptable spectroelectrochemical titrator: the midpoint reduction potential of the iron-sulfur center in lysine 2,3-aminomutase

Elaborations to an earlier design of an electron paramagnetic resonance (EPR) spectroelectrochemical titrator are described. While maintaining the anaerobic capabilities of the original design, a number of modifications and revisions have been introduced. The most significant modification is the use of a detachable spectral cell, making the apparatus modular and adaptable for multiple forms of spectroscopy. Additional modifications include removable reference, auxiliary, and working electrodes; modifications to facilitate sample transfer; and adaptations for operation within an anaerobic chamber. This apparatus has been used successfully in the coulometric titration of a [4Fe-4S] enzyme, as measured by EPR spectroscopy. The midpoint reduction potential for the 2+/1+ couple in the [4Fe-4S] cluster of lysine 2,3-aminomutase is -479+/-5mV, a value that falls within the range typical of ferredoxin-like iron-sulfur clusters.     

EPR determination of interaction redox potentials in a multiheme cytochrome: cytochrome c3 from Desulfovibrio desulfuricans Norway

In cytochromes c3 which contain four hemes per molecule, the redox properties of each heme may depend upon the redox state of the others. This effect can be described in terms of interaction redox potentials between the hemes and must be taken into account in the characterization of the redox properties of the molecule. We present here a method of measurement of these interactions based on the EPR study of the redox equilibria of the protein. The microscopic and macroscopic midpoint potentials and the interaction potentials are deduced from the analysis of the redox titration curves of the intensity and the amplitude of the EPR spectrum. This analysis includes a precise simulation of the spectrum of the protein in the oxidized state in order to determine the relative contribution of each heme to the spectral amplitude. Using our method on cytochrome c3 from D. desulfuricans Norway, we found evidence for the existence of weak interaction potentials between the hemes. The three interaction potentials which have been measured are characterized by absolute values lower than 20 mV in contrast with the values larger than 40-50 mV which have been reported for cytochrome c3 from D. gigas. Simulations of the spectra of samples poised at different potentials indicate a structural modification of the heme with the most negative potential during the first step of reduction. The correspondence between the redox sites as characterized by the EPR potentiometric titration and the hemes in the tridimensional structure is discussed.     

Assignment of individual heme EPR signals of Desulfovibrio baculatus (strain 9974) tetraheme cytochrome c3. A redox equilibria study

An EPR redox titration was performed on the tetraheme cytochrome c3 isolated from Desulfovibrio baculatus (strain 9974), a sulfate-reducer. Using spectral differences at different poised redox states of the protein, it was possible to individualize the EPR g-values of each of the four hemes and also to determine the mid-point redox potentials of each individual heme: heme 4 (-70 mV) at gmax = 2.93, gmed = 2.26 and gmin = 1.51; heme 3 (-280 mV) at gmax = 3.41; heme 2 (-300 mV) at gmax = 3.05, gmed = 2.24 and gmin = 1.34; and heme 1 (-355 mV) at gmx = 3.18. A previously described multi-redox equilibria model used for the interpretation of NMR data of D. gigas cytochrome c3 [Santos, H., Moura, J.J.G., Moura, I., LeGall, J. & Xavier, A. V. (1984) Eur. J. Biochem. 141, 283-296] is discussed in terms of the EPR results.     

Effect of redox potential on activity of hydrogenase 1 and hydrogenase 2 in Escherichia coli

This report elucidates the distinctions of redox properties between two uptake hydrogenases in Escherichia coli. Hydrogen uptake in the presence of mediators with different redox potential was studied in cell-free extracts of E. coli mutants HDK103 and HDK203 synthesizing hydrogenase 2 or hydrogenase 1, respectively. Both hydrogenases mediated H(2) uptake in the presence of high-potential acceptors (ferricyanide and phenazine methosulfate). H(2) uptake in the presence of low-potential acceptors (methyl and benzyl viologen) was mediated mainly by hydrogenase 2. To explore the dependence of hydrogen consumption on redox potential of media in cell-free extracts, a chamber with hydrogen and redox ( E(h)) electrodes was used. The mutants HDK103 and HDK203 exhibited significant distinctions in their redox behavior. During the redox titration, maximal hydrogenase 2 activity was observed at the E(h) below -80 mV. Hydrogenase 1 had maximum activity in the E(h) range from +30 mV to +110 mV. Unlike hydrogenase 2, the activated hydrogenase 1 retained activity after a fast shift of redox potential up to +500 mV by ferricyanide titration and was more tolerant to O(2). Thus, two hydrogenases in E. coli are complementary in their redox properties, hydrogenase 1 functioning at higher redox potentials and/or at higher O(2) concentrations than hydrogenase 2.     

Interconversions between the 3Fe and 4Fe forms of the iron-sulfur clusters in the ferredoxin from Thermodesulfobacterium commune: EPR characterization and potentiometric titration

The amount of 3Fe clusters in Thermodesulfobacterium commune ferredoxin is strongly dependent upon the presence of oxygen during the purification. An average of one 3Fe cluster per monomer can be found when the purification is not strictly anaerobic. These clusters are converted into |4Fe-4S| clusters by adding dithionite at usual pH and without adjunction of Fe²⁺. The EPR potentiometric titration reveals the existence of several types of 3Fe clusters with negative midpoint potentials differing by more than 100 mV. When the |4Fe-4S| clusters are partially reduced the EPR signal is composed of two different rhombic components. The component with g_z = 2.04 could be related to a site implicated in the interconversion processes. In the fully reduced state, the spectrum presents the typical features of two interacting |4Fe-4S| clusters as those observed in two |4Fe-4S| bacterial ferredoxins. From the redox titration curves the midpoint potentials of these clusters are estimated at −395 and −435 mV.     

Electricity production by Geobacter sulfurreducens attached to electrodes

Previous studies have suggested that members of the Geobacteraceae can use electrodes as electron acceptors for anaerobic respiration. In order to better understand this electron transfer process for energy production, Geobacter sulfurreducens was inoculated into chambers in which a graphite electrode served as the sole electron acceptor and acetate or hydrogen was the electron donor. The electron-accepting electrodes were maintained at oxidizing potentials by connecting them to similar electrodes in oxygenated medium (fuel cells) or to potentiostats that poised electrodes at +0.2 V versus an Ag/AgCl reference electrode (poised potential). When a small inoculum of G. sulfurreducens was introduced into electrode-containing chambers, electrical current production was dependent upon oxidation of acetate to carbon dioxide and increased exponentially, indicating for the first time that electrode reduction supported the growth of this organism. When the medium was replaced with an anaerobic buffer lacking nutrients required for growth, acetate-dependent electrical current production was unaffected and cells attached to these electrodes continued to generate electrical current for weeks. This represents the first report of microbial electricity production solely by cells attached to an electrode. Electrode-attached cells completely oxidized acetate to levels below detection (<10 micro M), and hydrogen was metabolized to a threshold of 3 Pa. The rates of electron transfer to electrodes (0.21 to 1.2 micro mol of electrons/mg of protein/min) were similar to those observed for respiration with Fe(III) citrate as the electron acceptor (E(o)' =+0.37 V). The production of current in microbial fuel cell (65 mA/m(2) of electrode surface) or poised-potential (163 to 1,143 mA/m(2)) mode was greater than what has been reported for other microbial systems, even those that employed higher cell densities and electron-shuttling compounds. Since acetate was completely oxidized, the efficiency of conversion of organic electron donor to electricity was significantly higher than in previously described microbial fuel cells. These results suggest that the effectiveness of microbial fuel cells can be increased with organisms such as G. sulfurreducens that can attach to electrodes and remain viable for long periods of time while completely oxidizing organic substrates with quantitative transfer of electrons to an electrode.     

Potentiometric analysis of the flavin cofactors of neuronal nitric oxide synthase

Midpoint reduction potentials for the flavin cofactors in the reductase domain of rat neuronal nitric oxide synthase (nNOS) in calmodulin (CaM)-free and -bound forms have been determined by direct anaerobic titration. In the CaM-free form, the FMN potentials are -49 +/- 5 mV (oxidized/semiquinone) -274 +/- 5 mV (semiquinone/reduced). The corresponding FAD potentials are -232 +/- 7, and -280 +/- 6 mV. The data indicate that each flavin can exist as a blue (neutral) semiquinone. The accumulation of blue semiquinone on the FMN is considerably higher than seen on the FAD due to the much larger separation (225 mV) of its two potentials (cf. 48 mV for FAD). For the CaM-bound form of the protein, the midpoint potentials are essentially identical: there is a small alteration in the FMN oxidized/semiquinone potential (-30 +/- 4 mV); the other three potentials are unaffected. The heme midpoint potentials for nNOS [-239 mV, L-Arg-free; -220 mV, L-Arg-bound; Presta, A., Weber-Main, A. M., Stankovich, M. T., and Stuehr, D. J. (1998) J. Am. Chem. Soc. 120, 9460-9465] are poised such that electron transfer from flavin domain is thermodynamically feasible. Clearly, CaM binding is necessary in eliciting conformational changes that enhance flavin to flavin and flavin to heme electron transfers rather than causing a change in the driving force.     

Pyrococcus furiosus glyceraldehyde 3-phosphate oxidoreductase has comparable W(6+/5+) and W(5+/4+) reduction potentials and unusual [4Fe-4S] EPR properties

Pyrococcus furiosus glyceraldehyde 3-phosphate oxidoreductase has been characterized using EPR-monitored redox titrations. Two different W signals were found. W(1)(5+) is an intermediate species in the catalytic cycle, with the midpoint potentials E(m)(W(6+/5+))=-507 mV and E(m)(W(5+/4+))=-491 mV. W(2)(5+) represents an inactivated species with E(m)(W(6+/5+))=-329 mV. The cubane cluster exhibits both S=3/2 and S=1/2 signals with the same midpoint potential: E(m)([4Fe-4S](2+/1+))=-335 mV. The S=1/2 EPR signal is unusual with all g values below 2.0. The titration results combined with catalytic voltammetry data are consistent with electron transfer from glyceraldehyde 3-phosphate first to the tungsten center, then to the cubane cluster and finally to the ferredoxin.     

Metabolism of carbon monoxide by Rhodopseudomonas gelatinosa: cell growth and properties of the oxidation system.

Rhodopseudomonas gelatinosa 1 grew as an anaerobic facultative methylotroph with carbon monoxide as the sole carbon and energy source. Carbon from CO was assimilated into cell material via the ribulose 1,5-bisphosphate carboxylase cycle. The CO oxidation system in R. gelatinosa was induced during growth with the gas substrate. Light-grown cells did not oxidize CO. Surprisingly, when strain 1 cells grown in the dark with CO were transferred to growth with both CO and light, they continued to use CO and then photometabolized after the CO gas flow was stopped. This change in the energy-yielding substrate resulted in a diauxic growth response. The use of CO in preference to light energy forms the basis of a system in the cells that controls photosynthetic differentiation. CO oxidation was assayed as CO-methyl viologen oxidoreductase. Methyl viologen reduction only occurred with CO; the dye was not reduced with other C1 compounds. In vitro methyl viologen was reduced best at 24 degrees C and at pH values above 8.5. Whole cells exhibited a Km of 12.5 microM for CO and a Vmax of 3,800 nmol of CO oxidized per mg of protein per min. This was a low-potential oxidation reaction that readily reduced the viologen dye triquat (1,1'-trimethylene-2,2'-dipyridilium dibromide) (E degrees' = -548 mV).     

Evidence for a proposed intermediate redox state in the CO/CO(2) active site of acetyl-CoA synthase (Carbon monoxide dehydrogenase) from Clostridium thermoaceticum

When samples of the enzyme in the C(red1) state were reduced with Ti(3+) citrate, the C-cluster stabilized in an EPR-silent state. Subsequent treatment with CO or dithionite yielded C(red2). The EPR-silent state formed within 1 min of adding Ti(3+) citrate, while C(red2) formed after 60 min. Ti(3+) citrate appeared to slow the rate by which C(red2) formed from C(red1) and stabilize the C-cluster in the previously proposed C(int) state. This is the first strong evidence for C(int), and it supports the catalytic mechanism that required its existence. This mechanism is analogous to those used by flavins and hydrogenases to convert between n = 2 and n = 1 processes. Ti(3+) citrate had a different effect on enzyme in a CO(2) atmosphere; it shifted reduction potentials of metal centers (relative to those obtained using CO) and did not stabilize C(int). Different redox behavior was also observed when methyl viologen and benzyl viologen were used as reductants. This variability was exploited to prepare enzyme samples in which EPR from C(red2) was present without interfering signals from B(red). The saturation properties of B(red) depended upon the redox state of the enzyme. Three saturation "modes", called Sat1-Sat3, were observed. Sat1 was characterized by a sharp g = 1.94 resonance and low-intensity g = 2. 04 and 1.90 resonances, and was observed in samples poised at slightly negative potentials. Sat2 was characterized by weak intensity from all three resonances, and was strictly associated with intermediate redox states and the presence of CO(2). Sat3 was characterized by strong broad resonances with normalized intensities essentially unchanged relative to nonsaturating conditions, and was observed at the most negative potentials. Each mode probably reflects different spatial relationships among magnetic components in the enzyme.     

A temperature-controlled, anaerobic cell for direct electrochemical studies.

The construction and operation of a cell for temperature-controlled, direct electrochemical studies of oxygen-sensitive materials are described. The borosilicate cell contains a pyrolytic graphite working electrode, a Ag/AgCl reference electrode, and a platinum counter electrode, all of which can be readily interchanged with other types of electrodes. It is surrounded by a water jacket constructed of steel and Lexan, which can easily maintain temperatures between 4 degrees C and at least 90 degrees C. The entire cell was designed to minimize the number, complexity, and expense of components, as well as minimize required sample volume (250 microliters) and sources of oxygen leakage. As examples of the cell's utility, the redox properties of two common organic redox dyes, methyl viologen and thionin, were determined by differential pulse voltammetry at temperatures from 30 to 90 degrees C.     

Redox characteristics of the tungsten DMSO reductase of Rhodobacter capsulatus

The dimethylsulfoxide reductase (DMSOR) from Rhodobacter capsulatus is known to retain its three-dimensional structure and enzymatic activity upon substitution of molybdenum, the metal that occurs naturally at the active site, by tungsten. The redox properties of tungsten-substituted DMSOR (W-DMSOR) have been investigated by a dye-mediated reductive titration with the concentration of the W(V) state monitored by EPR spectroscopy. At pH 7.0, E(m)(W(VI)/W(V)) is -194 mV and E(m)(W(V)/W(IV)) is -134 mV. Each E(m) value of W-DMSOR is significantly lower (220 and 334 mV, respectively) than that of the corresponding couple of Mo-DMSOR. These redox potentials are consistent with the ability of Mo-DMSOR to catalyze both the reduction of DMSO to DMS and the back reaction, whereas W-DMSOR is very effective in catalyzing the forward reaction, but shows no ability to catalyze the oxidation of DMS to DMSO.     

Investigation of the redox centres of periplasmic selenate reductase from Thauera selenatis by EPR spectroscopy

Periplasmic SER (selenate reductase) from Thauera selenatis is classified as a member of the Tat (twin-arginine translocase)-translocated (Type II) molybdoenzymes and comprises three subunits each containing redox cofactors. Variable-temperature X-band EPR spectra of the purified SER complex showed features attributable to centres [3Fe-4S]1+, [4Fe-4S]1+, Mo(V) and haem-b. EPR-monitored redox-potentiometric titration of the SerABC complex (SerA-SerB-SerC, a hetero-trimetric complex of alphabetagamma subunits) revealed that the [3Fe-4S] cluster (FS4, iron-sulfur cluster 4) titrated as n=1 Nernstian component with a midpoint redox potential (E(m)) of +118+/-10 mV for the [3Fe-4S]1+/0 couple. A [4Fe-4S]1+ cluster EPR signal developed over a range of potentials between 300 and -200 mV and was best fitted to two sequential Nernstian n=1 curves with midpoint redox potentials of +183+/-10 mV (FS1) and -51+/-10 mV (FS3) for the two [4Fe-4S]1+/2+ cluster couples. Upon further reduction, the observed signal intensity of the [4Fe-4S]1+ cluster decreases. This change in intensity can again be fitted to an n=1 Nernstian component with a midpoint potential (E(m)) of about -356 mV (FS2). It is considered likely that, at low redox potential (E(m) less than -300 mV), the remaining oxidized cluster is reduced (spin S=1/2) and strongly spin-couples to a neighbouring [4Fe-4S]1+ cluster rendering both centres EPR-silent. The involvement of both [3Fe-4S] and [4Fe-4S] clusters in electron transfer to the active site of the periplasmic SER was demonstrated by the re-oxidation of the clusters under anaerobic selenate turnover conditions. Attempts to detect a high-spin [4Fe-4S] cluster (FS0) in SerA at low temperature (5 K) and high power (100 mW) were unsuccessful. The Mo(V) EPR recorded at 60 K, in samples poised at pH 6.0, displays principal g values of g3 approximately 1.999, g2 approximately 1.996 and g1 approximately 1.965 (g(av) 1.9867). The dominant features at g2 and g3 are not split, but hyperfine splitting is observed in the g1 region of the spectrum and can be best simulated as arising from a single proton with a coupling constant of A1 (1H)=1.014 mT. The presence of the haem-b moiety in SerC was demonstrated by the detection of a signal at g approximately 3.33 and is consistent with haem co-ordinated by methionine and lysine axial ligands. The combined evidence from EPR analysis and sequence alignments supports the assignment of the periplasmic SER as a member of the Type II molybdoenzymes and provides the first spectro-potentiometric insight into an enzyme that catalyses a key reductive reaction in the biogeochemical selenium cycle.     

Studies on the Circular Dichroism (CD) and Electron Paramaguetic Resonance(EPR) Spe-ctroscopy of the Partially Oxidized FeMo Protein from Azotobacter vinelandii Nitrogenase

After nitrogenase FeMo protein from A. vinelandii was incubated with a large excess (5–6 equivalents) of indigo carmine for 30–60 min., all of P-clusters in the partially oxidized FeMo protein were oxidized, but all of FeMoCo in the protein were still in the reductive state. The oxidized P-clusters in the protein were able to be completely reduced by Na2S2O4(DT) and the reduction was accelerated by methyl viologen (MV). And all of FeMoCo in the protein were firstly oxidized by some oxidants such as methylene blue. The numbers of the redox equivalent of P-cluster and FeMoCo have been obtained by the CD reductive titration of and EPR/ ABS time course on the oxidation of the partially oxidizeA FeMo protein, respectivelly.     

Thermodynamic basis of electron transfer in dihydroorotate dehydrogenase B from Lactococcus lactis: analysis by potentiometry, EPR spectroscopy, and ENDOR spectroscopy

Dihydroorotate dehydrogenase B (DHODB) is a complex iron-sulfur flavoprotein that catalyzes the conversion of dihydroorotate to orotate and the reduction of NAD(+). The enzyme is a dimer of heterodimers containing an FMN, an FAD, and a 2Fe-2S center. UV-visible, EPR, and ENDOR spectroscopies have been used to determine the reduction potentials of the flavins and the 2Fe-2S center and to characterize radicals and their interactions. Reductive titration using dithionite indicates a five-electron capacity for DHODB. The midpoint reduction potential of the 2Fe-2S center (-212 +/- 3 mV) was determined from analysis of absorption data at 540 nm, where absorption contributions from the two flavins are small. The midpoint reduction potentials of the oxidized/semiquinone (E(1)) and semiquinone/hydroquinone (E(2)) couples for the FMN (E(1) = -301 +/- 6 mV; E(2) = -252 +/- 8 mV) and FAD (E(1) = -312 +/- 6 mV; E(2) = -297 +/- 5 mV) were determined from analysis of spectral changes at 630 nm. Corresponding values for the midpoint reduction potentials for FMN (E(1) = -298 +/- 4 mV; E(2) = -259 +/- 5 mV) in the isolated catalytic subunit (subunit D, which lacks the 2Fe-2S center and FAD) are consistent with the values determined for the FMN couples in DHODB. During reductive titration of DHODB, small amounts of the neutral blue semiquinone are observed at approximately 630 nm, consistent with the measured midpoint reduction potentials of the flavins. An ENDOR spectrum of substrate-reduced DHODB identifies hyperfine couplings to proton nuclei similar to those recorded for the blue semiquinone of free flavins in aqueous solution, thus confirming the presence of this species in DHODB. Spectral features observed during EPR spectroscopy of dithionite-reduced DHODB are consistent with the midpoint reduction potentials determined using UV-visible spectroscopy and further identify an unusual EPR signal with very small rhombic anisotropy and g values of 2.02, 1.99, and 1.96. This unusual signal is assigned to the formation of a spin interacting state between the FMN semiquinone species and the reduced 2Fe-2S center. Reduction of DHODB using an excess of NADH or dihydroorotate produces EPR spectra that are distinct from those produced by dithionite. From potentiometric studies, the reduction of the 2Fe-2S center and the reduction of the FMN occur concomitantly. The study provides a detailed thermodynamic framework for electron transfer in this complex iron-sulfur flavoprotein.     

Electricity Production by Geobacter sulfurreducens Attached to Electrodes

Previous studies have suggested that members of the Geobacteraceae can use electrodes as electron acceptors for anaerobic respiration. In order to better understand this electron transfer process for energy production, Geobacter sulfurreducens was inoculated into chambers in which a graphite electrode served as the sole electron acceptor and acetate or hydrogen was the electron donor. The electron-accepting electrodes were maintained at oxidizing potentials by connecting them to similar electrodes in oxygenated medium (fuel cells) or to potentiostats that poised electrodes at +0.2 V versus an Ag/AgCl reference electrode (poised potential). When a small inoculum of G. sulfurreducens was introduced into electrode-containing chambers, electrical current production was dependent upon oxidation of acetate to carbon dioxide and increased exponentially, indicating for the first time that electrode reduction supported the growth of this organism. When the medium was replaced with an anaerobic buffer lacking nutrients required for growth, acetate-dependent electrical current production was unaffected and cells attached to these electrodes continued to generate electrical current for weeks. This represents the first report of microbial electricity production solely by cells attached to an electrode. Electrode-attached cells completely oxidized acetate to levels below detection (<10 μM), and hydrogen was metabolized to a threshold of 3 Pa. The rates of electron transfer to electrodes (0.21 to 1.2 μmol of electrons/mg of protein/min) were similar to those observed for respiration with Fe(III) citrate as the electron acceptor (E^o′ =+0.37 V). The production of current in microbial fuel cell (65 mA/m² of electrode surface) or poised-potential (163 to 1,143 mA/m²) mode was greater than what has been reported for other microbial systems, even those that employed higher cell densities and electron-shuttling compounds. Since acetate was completely oxidized, the efficiency of conversion of organic electron donor to electricity was significantly higher than in previously described microbial fuel cells. These results suggest that the effectiveness of microbial fuel cells can be increased with organisms such as G. sulfurreducens that can attach to electrodes and remain viable for long periods of time while completely oxidizing organic substrates with quantitative transfer of electrons to an electrode.     

On the redox properties of three bisulfite reductases from the sulfate-reducing bacteria.

The redox properties of purified bisulfite reductases from Desulfovibrio gigas, D. desulfuricans (Norway) and Desulfotomaculum ruminis, containing non-heme iron and siroheme have been studied by EPR spectroscopy. Each enzyme shows ferric siroheme EPR signals which are not completely reduced by dithionite after 20 min, but are readily reduced within 1 min by dithionite plus methyl viologen. With the latter reducing system, each reductase also reveals a variable Beinert “g=1.94” type iron-sulfur signal. Reaction of each reductase with reduced methyl viologen results in reduction of only the siroheme. These results suggest different redox potentials for the iron-sulfur and siroheme moieties, and indicate that their functional properties are similar for each reductase.     

Preferential use of an anode as an electron acceptor by an acidophilic bacterium in the presence of oxygen

Several anaerobic metal-reducing bacteria have been shown to be able to donate electrons directly to an electrode. This property is of great interest for microbial fuel cell development. To date, microbial fuel cell design requires avoiding O(2) diffusion from the cathodic compartment to the sensitive anodic compartment. Here, we show that Acidiphilium sp. strain 3.2 Sup 5 cells that were isolated from an extreme acidic environment are able to colonize graphite felt electrodes. These bacterial electrodes were able to produce high-density electrocatalytic currents, up to 3 A/m(2) at a poised potential of +0.15 V (compared to the value for the reference standard calomel electrode) in the absence of redox mediators, by oxidizing glucose even at saturating air concentrations and very low pHs.     

An electrochemical thin-layer cell for spectroscopic studies of photosynthetic electron-transport components

An electrochemical thin-layer cell, 0.5 ml in volume and 0.25 mm in pathlength, employing an optically transparent gold minigrid electrode has been constructed and used in redox studies of photosynthetic electron-transport components. Light-induced absorption changes accompanying P700 photo-oxidation in photosystem-I subchloroplasts were measured at a series of electrochemically poised potentials at 88°K. The fluorescence-yield changes in photosystem-II subchloroplasts were measured as the poised potential was varied electrochemically. The utility of the cell was further demonstrated by a redox titration of soluble (spinach) ferredoxin using circular dichroism to monitor changes in the redox state. One important advantage of electrochemical vs chemical titration of photosynthetic electron-transport components is that it is possible to attain much more negative potentials than is thermodynamically possible in chemical titrations. This permits the exhaustive titration of certain components at physiologically reasonable pH values not possible chemically.     

EPR spectroscopy of the iron-sulphur cluster and sirohaem in the dissimilatory sulphite reductase (desulphoviridin) from Desulphovibrio gigas.

Desulphoviridin in the oxidized state showed EPR signals around g = 6, consistent with the sirohaem being in the high-spin ferric state. This was unreactive with sulphite, sulphide or cyanide; but readily reduced by methyl viologen. When the enzyme was treated with Na2S2O4 the sirohaem was slowly reduced and a spectrum of a reduced iron-sulphur cluster at g = 2.07, 1.93, 1.91 appeared over the course of an hour. An intermediate in this reaction was indicated by a free radical signal which appeared within seconds and then gradually disappeared. On treatment with nitrite and reduced methyl viologen, the enzyme gave a spectrum of a nitroxide derivative similar to that seen with plant nitrite reductase. The midpoint reduction potential of the haem was estimated to be -310 mV or less. The iron-sulphur cluster has a very low potential, being only reduced in the presence of free Na2S2O4 around -560 mV. Desulphoviridin can be classed with sirohaem-containing iron-sulphur proteins.