Iron-sulfur proteins of the green photosynthetic bacterium Chlorobium.

The iron-sulfur proteins of the green photosynthetic bacterium Chlorobium have been characterized by oxidation-reduction potentiometry in conjunction with low-temperature electron paramagnetic resonance spectroscopy. Chlorobium ferredoxin was the only iron-sulfur protein detected in the soluble fraction; no high-potential iron-sulfur protein was observed. In addition, high-potential iron-sulfur protein was not detected in the chromatophores. Four chromatophore-bound iron-sulfur proteins were detected. One is the "Rieske" type iron-sulfur protein with a g-value of 1.90 in the reduced state; the protein has a midpoint potential of + 160 mV (pH 7.0), and this potential is pH dependent. Three g=1.94 chromatophore-bound iron-sulfur proteins were observed, with midpoint potentials of -25, -175, and about -550 mV. A possible role for the latter iron-sulfur protein in the primary photochemical reaction in Chlorobium is considered.     

Thermodynamic resolution of the iron-sulfur centers of the succinic dehydrogenase of Rhodopseudomonas sphaeroides.

A single alkaline wash removes most of the succinic dehydrogenase activity from chromatophores of Rhodopseudomonas sphaeroides. Three iron-sulfur centers are also removed by this washing. Two of these are ferredoxin-like centers with electron paramagnetic resonance signals at g_v = 1.94 and midpoint potentials of +50 and ?250 mV at pH 7. The third is a high-potential iron-sulfur protein type signal centered at g 2.01 and a midpoint potential of +80 mV at pH 7. These centers have very similar properties to those of the well-characterized mammalian succinic dehydrogenase and account for the majority of iron-sulfur centers observed in chromatophores. Because it is so easily removed, it is concluded that succinic dehydrogenase is located on the outer surface of the chromatophore membrane, a conclusion supported by the fact that removal of the enzyme does not interfere with the kinetics of light-induced electron flow, nor does it allow cytochrome c₂ to escape from inside the chromatophore vesicles.     

Characterization of the iron-sulfur protein of the mitochondrial outer membrane partially purified from beef kidney cortex

The iron-sulfur protein present in the mitochondrial outer membrane has been partially purified from beef kidney cortex mitochondria be means of selective solubilization followed by DEAE-cellulose chromatography. The EPR spectrum of the iron-sulfur protein with g-values at 2.01, 1.94 and 1.89 was well resolved up to 200 K which is unusual for an iron-sulfur protein. Analyses confirmed a center with two iron and two labile sulfur atoms in the protein. By measuring the effect of oxidation-reduction potential on the EPR signal amplitude, midpoint potentials at pH 7.2 were determined both for the purified ironsulfur protein, +75 (±5) mV, and in prepared mitochondrial outer membrane, +62 (±6) mV. At pH 8.2 slightly lower values were indicated, +62 and 52 mV, respectively. The oxidation-reduction equilibrium involved a one electron transfer. A functional relationship to the rotenone-insensitive NADH-cytochrome c oxidoreductase in the mitochondrial outer membrane is suggested. Both this activity and the iron-sulfur center were sensitive to acidities slightly below pH 7 in contrast to the iron-sulfur centers of the inner membrane.     

Electron spin resonance characterization of Chromatium D hemes,non-heme irons and the components involved in primary photochemistry

The combination of redox potentiometry with low temperature electron spin resonance (ESR) spectroscopy has led to further characterization of electron transfer components of Chromatium D. These include the readily buffer-soluble cytochromes c₅₅₃ and c′ and the high-potential iron-sulfur protein in the isolated state and associated with the chromatophore membrane. Buffer-insoluble cytochrome c₅₅₃, cytochro—me c₅₅₅, bacteriochlorophyll and the primary electron acceptor have been characterized both in the chromatophore membrane and also in a sodium dodecylsulfate detergent-solubilized subchromatophore preparation. Two iron-sulfur proteins have been revealed which are present in the chromatophore membrane but are released on treatment with sodium dodecylsulfate. They have central g values at 1.90 and 1.94 and have estimated midpoint potentials at pH 7.4 (E_m7·4) at +280 mV and ?100 mV, respectively, when associated with the chromatophore.In the membrane associated state the apparent E_m of cytochrome c′ is approximately 200 mV more positive than the E_m values reported for the free state; this implies either that the reduced form of cytochrome c′ binds to the membrane (or to a component therein) to a degree which is > 10³ times greater than that of the oxidized form or that the E_m shift results from membrane solvation. In the case of the high-potential iron-sulfur protein however, its E_m when associated with the chromatophore membrane is similar to that reported in the isolated state. The light-induced oxidation of the high-potential iron-sulfur protein at room temperature appears to be linked only to the oxidation of cytochrome c₅₅₅; it could serve as an electron pool in equilibrium with cytochrome c₅₅₅ in the cyclic electron flow system.The redox component defined in the reduced state by its g_y = 1.82 and g_x = 1.62 ESR spectrum satisfies the following criteria for its identification as the primary electron acceptor of P883. (a) The E_m7·4 value of the g = 1.82 component is ?120 ± 25mV. (b) At ?70 mV, where the g = 1.82 component is mainly oxidized in the dark, brief illumination at low temperature which causes the irreversible oxidation of one cytochrome c₅₅₃ heme, also induces the permanent reduction of the g = 1.82 component; the extent of reduction after brief illumination, given by the g = 1.82 signal height, is the same as that induced chemically at ?270 mV showing it to be fully reduced by the receipt of a single electron. (c) At more positive potentials where cytochrome c₅₅₃ is oxidized and is not involved in low-temperature reactions, the light-induced low-temperature kinetics of the g = 1.82 signal are reversible; the flash-induced g = 1.82 formation and subsequent dark decay are the same as those for the flash-induced P⁺883 (g = 2) formation and dark decay. We suggest that until a full physical-chemical characterization is completed this g = 1.82 component be designated “photoredoxin”.     

Identification of a g equals 1.90 high-potential iron-sulfur protein in chloroplasts.

A new bound iron-sulfur protein has been identified in spinach chloroplasts. In the reduced form, this protein has an electron paramagnetic resonance spectrum at 20°K with g-values of 2.02 and 1.90. The midpoint oxidation-reduction potential (E_m) of the protein, which is pH-independent, is +290 mV. These properties are similar to those of the “Rieske” g = 1.90 iron-sulfur protein of mitochondrial Complex III.     

Characterization of iron-sulfur clusters in rat liver submitochondrial particles by electron paramagnetic resonance spectroscopy. Alterations produced by chronic ethanol consumption

Iron-sulfur clusters present in rat liver submitochondrial particles were characterized by ESR at temperatures between 30 and 5.5 K combined with potentiometric titrations. The spectral and thermodynamic characteristics of the iron-sulfur clusters were generally similar to those previously reported for pigeon or bovine heart submitochondrial particles. Clusters N-1a, N-1b, N-2, N-3 and N-4 of NADH dehydrogenase had midpoint oxidation-reduction potentials at pH 7.5 of ?425, ?265, ?85, ?240 and ?260 mV, respectively. Clusters S-1 and S-3 of succinate dehydrogenase had midpoint potentials of 0 and +65 mV, respectively. The iron-sulfur cluster of electron-transferring flavoprotein-ubiquinone oxidoreductase exhibited the g_z signal at g = 2.08 and had a midpoint potential of +30 mV. This signal was relatively prominent in rat liver compared to pigeon or bovine heart.Submitochondrial particles from rats chronically treated with ethanol (36% of total calories, 40 days) showed decreases of 20–30% in amplitudes of signals due to clusters N-2, N-3 and N-4 compared to those from pair-fed control rats. Signals from clusters N-1b, S-1, S-3 and electron-transferring flavoprotein-ubiquinone oxidoreductase were unaffected. Microwave power-saturation behavior was similar for both submitochondrial particle preparations, suggesting that the lower signal amplitudes reflected a lower content of these particular clusters. NADH dehydrogenase activity was significantly decreased (46%), whilst succinate dehydrogenase activity was elevated (25%), following chronic ethanol consumption. The results indicate that chronic ethanol treatment leads to an alteration of the structure and function of the NADH dehydrogenase segment of the electron transfer chain. This alteration is one of the factors contributing to the lower respiration rates observed following chronic ethanol administration.     

Thermodynamic and EPR characterization of mitochondrial succinate-cytochrome c reductase-phospholipid complexes

Succinate-cytochrome c reductase can be easily solubilized in a phospholipid mixture (1:1, lysolecithin:lecithin) in the absence of detergents. The resulting solution contains two b cytochromes with half-reduction potentials of 95 ± 10 mV (b₅₆₁), and 0 ± 10 mV (b₅₆₆) and cytochrome c₁ (E_{m 7.2} = +280±5 mV). The oxidation-reduction midpoint potentials obtained by optical potentiometric titrations are identical to those determined by the EPR titrations and are 40–60 mV higher than the corresponding midpoint potentials of these cytochromes in intact mitochondria. In contrast to detergent-suspended preparations, no CO-sensitive cytochrome b can be detected in the phospholipid-solubilized preparation or intact mitochondria. The half-reduction potential of cytochrome b₅₆₆ is pH-dependent above pH 7.0 (?60 mV/pH unit) while that of b₅₆₁ is essentially pH-independent from pH 6.7–8.5, in contrast to its pH dependence in intact mitochondria. EPR characterizations show the presence of three oxidized low-spin heme-iron signals with g values of 3.78, 3.41 and 3.37. The identification of these signals with cytochromes b₅₆₆ (b_T), b₅₆₁ (b_K) and c₁ respectively is made on the basis of redox midpoint potentials. No significant amounts of oxidized high-spin heme-iron are detectable. In addition, the preparation contains four distinct types of iron-sulfur centers: S₁ and S₂ (E_{m 7.4} = ?260 mV and 0 mV), and two iron-sulfur proteins which are associated with the cytochrome b-c₁ complex: Rieske's iron-sulfur protein (E_{m 7.4} = +280 mV) and Ohnishi's Center 5 (E_{m 7.4} = +35 mV).     

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.     

Redox titration of electron acceptor Q and the plastoquinone pool in Photosystem II

The primary photochemical quencher Q and the secondary electron acceptor pool in Photosystem II have been titrated. We used particles of Scenedesmus mutant No. 8 that lack System I and allowed the system to equilibrate with external redox mediators in darkness prior to measurement of the fluorescence rise curve.The titration of Q, as indicated by the dark level of F_i, occurs in two discrete steps. The high-potential component (Q_h) has a midpoint potential of +68 mV (pH 7.2) and accounts for ～67% of Q. The pH sensitivity of the midpoint potential is ?60 mV, indicating the involvement of 1 $\text{H}{}^{\mathrm{+}}\text{e}$. The low-potential component (Q₁) accounts for the remaining 33% of Q and shows a midpoint potential near?300 mV (pH 7.2).The plastoquinone pool, assayed as the half-time of the fluorescence rise curve, titrates as a single component with a midpoint potential 30–40 mV more oxidizing than that of Q_h, i.e., at 106 mV (pH 7.2). The E_m shows a pH sensitivity of ?60 mV/pH unit, indicating the involvement of 1 $\text{H}{}^{\mathrm{+}}\text{e}$. The observation that all 12–14 electron equivalents in the pool titrate as a single component indicates that the heterogeneity otherwise observed in the secondary acceptor system is a kinetic rather than a thermodynamic property.Illumination causes peculiar, and as yet unclarified, changes of both Q and the secondary pool under anaerobic conditions that are reversed by oxygen.     

Isolation and characterization of two soluble heme c-containing proteins from Chromatium vinosum

The photosynthetic purple sulfur bacterium Chromatium vinosum has been shown to possess two previously undetected heme c-containing, soluble proteins. One is an acidic, c-type cytochrome with a molecular weight of 12 300 and an oxidation-reduction midpoint potential (at pH 8.0) of ?82 mV. The other protein is a basic protein with a molecular weight of 11 900 and an oxidation-reduction midpoint potential (at pH 8.0) of ?110 mV. The basic protein, in both oxidized and reduced forms, has optical spectra similar to those of myoglobin and the oxidized C. vinosum protein exhibits a high-spin heme EPR spectrum similar to that of metmyoglobin. Furthermore, the basic C. vinosum protein binds CO and O₂. The spectra of the CO and O₂ complexes show significant similarities with the respective myoglobin complexes. Possible functions for an O₂-binding protein in C. vinosum are discussed.     

The influence of a surface charge on the electronic and steric structure of a high potential iron-sulfur protein

The recombinant high-potential iron-sulfur protein (HiPIP) iso-I from Ectothiorhodospira halophila has been mutated at position 68. The αC of Val 68 is within a 0.6-nm sphere from the closest iron ion of the cluster. The valine residue has been replaced by a negatively charged glutamate residue (V68E) and by a positively charged lysine residue (V68K). With respect to the recombinant wild-type protein the reduction potentials of the V68E and V68K variants are –21±2 and +29±2?mV respectively (200?mM NaCl, pH?7, 25??°C). The solution structure of the V68E mutant was solved up to a pairwise RMSD of 66?pm for backbone atoms and 138?pm for all heavy atoms. The structure of the variant is very similar to that of recombinant wild type, indicating that the observed changes in reduction potentials are largely due to the effect of the introduced charges. It is proposed that the valence distribution within the oxidized iron-sulfur cluster is affected only slightly by the change in charge at position 68, but consistently with a simple electrostatic model.     

Oxidation-reduction potentials of cytochromes P-450 and ferredoxin in the bovine adrenal: Their modification by substrates and inhibitors

The midpoint reduction potentials of the haem iron in bovine adrenal cytochrome P-450 and its associated iron-sulphur protein, adrenal ferredoxin, have been measured, using EPR spectroscopy to monitor the high and low spin ferric haem iron and reduced adrenal ferredoxin signals as a function of potential, in mitochondrial and microsomal suspensions.In mitochondria the high spin (substrate-bound) cytochrome P-450 showed single-component one-electron plots under most conditions; at pH 6.65 cholesterol side-chain cleavage cytochrome P-450 (P-450_scc) had a midpoint E_m = ?305 mV; at pH 8.0 11β-hydroxylase cytochrome P-450 (P-450_11β) had E_m = ?335 mV. Low spin cytochrome P-450 showed more complex titration curves under all conditions, which could be most simply interpreted in terms of two one-electron components with midpoint potentials approx. ?360 and ?470 mV, with varying intensities. During treatments that caused substrate binding, only the ?470 mV component was reduced in magnitude. On sonication and removal of adrenal ferredoxin, the ?470 mV low spin component was converted to higher potential. The potentials could also be altered by the cytochrome P-450 inhibitors aminoglutethimide and metyrapone. In the microsomes, a high spin component of cytochrome P-450 (E_m ≈ ?290 mV) was observed even at pH 8.0, suggesting the binding of an endogenous substrate, while the low spin P-450 showed a predominance of the ?360 mV component. The midpoint potential of membrane-bound adrenal ferredoxin under these various conditions was found to be ?248 mV ± 15 mV.     

Nonredundant Roles for Cytochrome c2 and Two High-Potential Iron-Sulfur Proteins in the Photoferrotroph Rhodopseudomonas palustris TIE-1

The purple bacterium Rhodopseudomonas palustris TIE-1 expresses multiple small high-potential redox proteins during photoautotrophic growth, including two high-potential iron-sulfur proteins (HiPIPs) (PioC and Rpal_4085) and a cytochrome c₂. We evaluated the role of these proteins in TIE-1 through genetic, physiological, and biochemical analyses. Deleting the gene encoding cytochrome c₂ resulted in a loss of photosynthetic ability by TIE-1, indicating that this protein cannot be replaced by either HiPIP in cyclic electron flow. PioC was previously implicated in photoferrotrophy, an unusual form of photosynthesis in which reducing power is provided through ferrous iron oxidation. Using cyclic voltammetry (CV), electron paramagnetic resonance (EPR) spectroscopy, and flash-induced spectrometry, we show that PioC has a midpoint potential of 450 mV, contains all the typical features of a HiPIP, and can reduce the reaction centers of membrane suspensions in a light-dependent manner at a much lower rate than cytochrome c₂. These data support the hypothesis that PioC linearly transfers electrons from iron, while cytochrome c₂ is required for cyclic electron flow. Rpal_4085, despite having spectroscopic characteristics and a reduction potential similar to those of PioC, is unable to reduce the reaction center. Rpal_4085 is upregulated by the divalent metals Fe(II), Ni(II), and Co(II), suggesting that it might play a role in sensing or oxidizing metals in the periplasm. Taken together, our results suggest that these three small electron transfer proteins perform different functions in the cell.     

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.     

Redox potential of plastoquinone A in spinach chloroplasts

The redox potential of plastoquinone A in spinach chloroplasts was determined. The midpoint potential of the quinone is about +80 mV at pH 7.0 with an n value of 2. The pH-dependence of the potential is ?30 mV per pH between pH 4.0 and 5.7, and ?60 mV per pH between pH 5.7 and 8.0. The change of the slope at pH 5.7 is interpreted as the protonation of the oxidized plastoquinone A.     

Identification of a stable ubisemiquinone and characterization of the effects of ubiquinone oxidation-reduction status on the Rieske iron-sulfur protein in the three-subunit ubiquinol-cytochrome c oxidoreductase complex of Paracoccus denitrificans

The ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex from Paracoccus denitrificans exhibits a thermodynamically stable ubisemiquinone radical detectable by EPR spectroscopy. The radical is centered at g = 2.004, is sensitive to antimycin, and has a midpoint potential at pH 8.5 of +42 mV. These properties are very similar to those of the stable ubisemiquinone (Qi) previously characterized in the cytochrome bc1 complexes of mitochondria. The micro-environment of the Rieske iron-sulfur cluster in the Paracoccus cytochrome bc1 complex changes in parallel with the redox state of the ubiquinone pool. This change is manifested as shifts in the gx, gy, and gz values of the iron-sulfur cluster EPR signal from 1.80, 1.89, and 2.02 to 1.76, 1.90, and 2.03, respectively, as ubiquinone is reduced to ubiquinol. The spectral shift is accompanied by a broadening of the signal and follows a two electron reduction curve, with a midpoint potential at pH 8.5 of +30 mV. A hydroxy analogue of ubiquinone, UHDBT, which inhibits respiration in the cytochrome bc1 complex, shifts the gx, gy, and gz values of the iron-sulfur cluster EPR signal to 1.78, 1.89, and 2.03, respectively, and raises the midpoint potential of the iron-sulfur cluster at pH 7.5 from +265 to +320 mV. These changes in the micro-environment of the Paracoccus Rieske iron-sulfur cluster are like those elicited in mitochondria. These results indicate that the cytochrome bc1 complex of P. denitrificans has a binding site for ubisemiquinone and that this site confers properties on the bound ubisemiquinone similar to those in mitochondria. In addition, the line shape of the Rieske iron-sulfur cluster changes in response to the oxidation-reduction status of ubiquinone, and the midpoint of the iron-sulfur cluster increases in the presence of a hydroxyquinone analogue of ubiquinone. The latter results are also similar to those observed in the mitochondrial cytochrome bc1 complex. However, unlike the mitochondrial complexes, which contain eight to 11 polypeptides and are thought to contain distinct quinone binding proteins, the Paracoccus cytochrome bc1 complex contains only three polypeptide subunits, cytochromes b, c1, and iron-sulfur protein. The ubisemiquinone binding site and the site at which ubiquinone and/or ubiquinol bind to affect the Rieske iron-sulfur cluster in Paracoccus thus exist in the absence of any distinct quinone binding proteins and must be composed of domains contributed by the cytochromes and/or iron-sulfur protein.     

EPR and redox characterization of iron-sulfur centers in nitrate reductases A and Z from Escherichia coli. Evidence for a high-potential and a low-potential class and their relevance in the electron-transfer mechanism.

The redox properties of the iron-sulfur centers of the two nitrate reductases from Escherichia coli have been investigated by EPR spectroscopy. A detailed study of nitrate reductase A performed in the range +200 mV to -500 mV shows that the four iron-sulfur centers of the enzyme belong to two classes with markedly different redox potentials. The high-potential group comprises a [3Fe-4S] and a [4Fe-4S] cluster whose midpoint potentials are +60 mV and +80 mV, respectively. Although these centers are magnetically isolated, they are coupled by a significant anticooperative redox interaction of about 50 mV. The [4Fe-4S]1+ center occurs in two different conformations as shown by its composite EPR spectrum. The low-potential group contains two [4Fe-4S] clusters with more typical redox potentials (-200 mV and -400 mV). In the fully reduced state, the three [4Fe-4S]1+ centers are magnetically coupled, leading to a broad featureless spectrum. The redox behaviour of the high-pH EPR signal given by the molybdenum cofactor was also studied. The iron-sulfur centers of the second nitrate reductase of E. coli, nitrate reductase Z, exhibit essentially the same characteristics than those of nitrate reductase A, except that the midpoint potentials of the high-potential centers appear negatively shifted by about 100 mV. From the comparison between the redox centers of nitrate reductase and of dimethylsulfoxide reductase, a correspondence between the high-potential iron-sulfur clusters of the two enzymes can be proposed.     

Iron-sulphur cluster composition and redox properties of two ferredoxins from Desulfovibrio desulfuricans Norway strain

Two ferredoxins from Desulfovibrio desulfuricans, Norway Strain, were investigated by EPR spectroscopy. Ferredoxin I appears to be a conventional [4Fe-4S]^2+;1+ ferredoxin, with a midpoint reduction potential of ?374 mV at pH 8. Ferredoxin II when reduced, at first showed a more complex spectrum, indicating an interaction between two [4Fe-4S] clusters, and probably, has two clusters per protein subunit. Upon reductive titration ferredoxin II changed to give a spectrum in which no intercluster interaction was seen. The midpoint potentials of the native and modified ferredoxin at pH 8 were estimated to be ?500 and ?440 mV, respectively.     

Physiochemical properties of ferralterin. A regulatory iron-sulfur protein functional in oxygenic photosynthesis

Ferralterin, an iron-sulfur protein identified earlier in chloroplasts and cyanobacteria, was purified to homogeneity from spinach leaves and Nostoc muscorum cells. When isolated from both sources, ferralterin showed a molecular weight of about 28,000 and was comprised of three subunits: one of molecular weight 12,000 and two, apparently identical, of molecular weight 7000. Based on the Lowry method of protein estimation, ferralterin contained approximately 3 g atoms each of nonheme iron and acid-labile sulfide per mole. The iron-sulfur cluster of ferralterin showed unusual redox and electron paramagnetic resonance (EPR) properties. Ferralterin was EPR silent as isolated and did not show an EPR signal on addition of reductants such as sodium dithionite or on exposure to illuminated chloroplast membranes. These reducing conditions also had no significant effect on the absorption spectrum of isolated ferralterin. The ferralterin iron-sulfur cluster was oxidized selectively by ferricyanide and showed a midpoint redox potential of +410 mV. Ferricyanide-oxidized ferralterin was characterized by a low-temperature EPR signal with g values of 2.10, 2.05, and 2.00 (spinach) and 2.09, 2.04, and 1.98 (Nostoc). When oxidized by ferricyanide, the iron-sulfur cluster could be reduced by a variety of reductants, including illuminated chloroplast membranes. The results are consistent with the conclusion that, like several other iron-sulfur enzymes (aconitase, glutamine phospho-ribosylpyrophosphate amidotransferase, hydrogenase), ferralterin achieves its catalytic effect via an active group independently of a redox change in the iron-sulfur chromophore.     

Characterization of the iron-sulfur protein of the mitochondrial outer membrane partially purified from beef kidney cortex.

The iron-sulfur protein present in the mitochondrial outer membrane has been partially purified from beef kidney cortex mitochondria by means of selective solubilization followed by DEAE-cellulose chromatography. The EPR spectrum of the iron-sulfur protein with g-values at 2.01, 1.94 and 1.89 was well resolved up to 200 K which is unusual for an iron-sulfur protein. Analyses confirmed a center with two iron and two labile sulfur atoms in the protein. By measuring the effect of oxidation-reduction potential on the EPR signal amplitude, midpoint potentials at pH 7.2 were determined both for the purified iron-sulfur protein, +75 (+/- 5) mV, and in prepared mitochondrial outer membrane, +62 (+/- 6) mV. At pH 8.2 slightly lower values were indicated, +62 and 52 mV, respectively. The oxidation-reduction equilibrium involved a one electron transfer. A functional relationship to the rotenone-insensitive NADH-cytochrome c oxidoreductase in the mitochondrial outer membrane is suggested. Both this activity and the iron-sulfur center were sensitive to acidities slightly below pH 7 in contrast to the iron-sulfur centers of the inner membrane.