Thermodynamic and EPR characterization of iron-sulfur centers in the NADH-ubiquinone segment of the mitochondrial respiratory chain in pigeon heart

Several iron-sulfur centers in the NADH-ubiquinone segment of the respiratory chain in pigeon heart mitochondria and in submitochondrial particles were analyzed by the combined application of cryogenic EPR (between 30 and 4.2 °K) and potentiometric titration.Center N-1 (iron-sulfur centers associated with NADH dehydrogenase are designated with the prefix “N”) resolves into two single electron titrations with E_{m 7.2} values of ?380±20 mV and ?240±20 mV (Centers N-1a and N-1b, respectively). Center N-1a exhibits an EPR spectrum of nearly axial symmetry with g_// = 2.03, g_⊥ = 1.94, while that of Center N-1b shows more apparent rhombic symmetry with g_z = 2.03, g_y = 1.94 and g_x = 1.91. Center N-2 also reveals EPR signals of axial symmetry at g_// = 2.05 and g_⊥ = 1.93 and its principal signal overlaps with those of Centers N-1a and N-1b. Center N-2 can be easily resolved from N-1a and N-1b because of its high E_{m 7.2} value (?20±20 mV).Resolution of Centers N-3 and N-4 was achieved potentiometrically in submitochondrial particles. The component with E_{m 7.2} = ? 240±20 mV is defined as Center N-3 (g_z = 2.10, (g_y = 1.93?), g_x = 1.87); the ?405±20 mV component as Center N-4 (g_z = 2.11, (g_y = 1.93?), g_x = 1.88). At temperatures close to 4.2 °K, EPR signals at g = 2.11, 2.06, 2.03, 1.93, 1.90 and 1.88 titrate with E_{m 7.2} = ?260±20 mV. The multiplicity of peaks suggests the presence of at least two different ironsulfur centers having similar E_{m 7.2} values (?260±20 mV); hence, tentatively assigned as N-5 and N-6.Consistent with the individual E_{m 7.2} values obtained, addition of succinate results in the partial reduction of Center N-2, but does not reduce any other centers in the NADH-ubiquinone segment of the respiratory chain. Centers N-2, N-1b, N-3, N-5 and N-6 become almost completely reduced in the presence of NADH, while Centers N-1a and N-4 are only slightly reduced in pigeon heart submitochondrial particles. In pigeon heart mitochondria, the E_{m 7.2} of Center N-4 lies much closer to that of Center N-3, so that resolution of the Center N-3 and N-4 spectra is not feasible in mitochondrial preparations. E_{m 7.2} values and EPR lineshapes for the other ironsulfur centers of the NADH-ubiquinone segment in the respiratory chain of intact mitochondria are similar to those obtained in submitochondrial particle preparations. Thus, it can be concluded that, in intact pigeon heart mitochondria, at least five iron-sulfur centers show E_{m 7.2} values around -250 mV; Center N-2 exhibits a high E_{m 7.2} (?20±20 mV), while Center N-1a shows a very low E_{m 7.2} (?380±20 mV).     

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^?.     

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.     

Electron-spin resonance studies of the bound iron-sulfur centers in Photosystem I II. Correlation of P-700 triplet production with urea/ferricyanide inactivation of the iron-sulfur clusters

Photosystem I charge separation in a subchloroplast particle isolated from spinach was investigated by electron spin resonance (ESR) spectroscopy following graduated inactivation of the bound iron-sulfur centers by urea-ferricyanide treatment. Previous work demonstrated a differential decrease in iron-sulfur centers A, B and X which indicated that center X serves as a branch point for parallel electron flow through centers A and B (Golbeck, J.H. and Warden, J.T. (1982) Biochim. Biophys. Acta 681, 77–84). We now show that during inactivation the disappearance of iron-sulfur centers A, B, and X correlates with the appearance of a spin-polarized triplet ESR signal with |D| = 279·10⁻⁴ cm⁻¹ and |E| = 39·10⁻⁴ cm⁻¹. The triplet resonances titrate with a midpoint potential of +380 ± 10 mV. Illumination of the inactivated particles results in the generation of an asymmetric ESR signal with g = 2.0031 and ΔH_pp = 1.0 mT. Deconvolution of the P-700⁺ contribution to this composite resonance reveals the spectrum of the putative primary acceptor species, A₀, which is characterized by g = 2.0033 ± 0.0004 and ΔH_pp = 1.0 ± 0.2 mT. The data presented in this report do not substantiate the participation of the electron acceptor A₁ in PS I electron transport, following destruction of the iron-sulfur cluster corresponding to center X. We suggest that A₁ is closely associated with center X and that this component is decoupled from the electron-transport path upon destruction of center X. The inability to photoreduce A₁ in reaction centers lacking a functional center X may result from alteration of the reaction center tertiary structure by the urea-ferricyanide treatment or from displacement of A₁ from its binding site.     

Xanthine dehydrogenase from Pseudomonas putida 86: specificity,oxidation–reduction potentials of its redox-active centers,and first EPR characterization

Xanthine dehydrogenase (XDH) from Pseudomonas putida 86, which was induced 65-fold by growth on hypoxanthine, was purified to homogeneity. It catalyzes the oxidation of hypoxanthine, xanthine, purine, and some aromatic aldehydes, using NAD⁺ as the preferred electron acceptor. In the hypoxanthine:NAD⁺ assay, the specific activity of purified XDH was 26.7 U (mg protein)⁻¹. Its activity with ferricyanide and dioxygen was 58% and 4%, respectively, relative to the activity observed with NAD⁺. XDH from P. putida 86 consists of 91.0 kDa and 46.2 kDa subunits presumably forming an α₄β₄ structure and contains the same set of redox-active centers as eukaryotic XDHs. After reduction of the enzyme with xanthine, electron paramagnetic resonance (EPR) signals of the neutral FAD semiquinone radical and the Mo(V) rapid signal were observed at 77 K. Resonances from FeSI and FeSII were detected at 15 K. Whereas the observable g factors for FeSII resemble those of other molybdenum hydroxylases, the FeSI center in contrast to most other known FeSI centers has nearly axial symmetry. The EPR features of the redox-active centers of P. putida XDH are very similar to those of eukaryotic XDHs/xanthine oxidases, suggesting that the environment of each center and their functionality are analogous in these enzymes. The midpoint potentials determined for the molybdenum, FeSI and FAD redox couples are close to each other and resemble those of the corresponding centers in eukaryotic XDHs.     

The role of the Rieske iron-sulfur center as the electron donor to ferricytochrome c2 in Rhodopseudomonas sphaeroides

The Rieske iron-sulfur center in the photosynthetic bacterium Rhodopseudomonas sphaeroides appears to be the direct electron donor to ferricytochrome c₂, reducing the cytochrome on a submillisecond timescale which is slower than the rapid phase of cytochrome oxidation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{3–5 μ}\text{s}$). The reduction of the ferricytochrome by the Rieske center is inhibited by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) but not by antimycin. The slower (1–2 ms) antimycin-sensitive phase of ferricytochrome c₂ reduction, attributed to a specific ubiquinone-10 molecule (Q_z), and the associated carotenoid spectral response to membrane potential formation are also inhibited by UHDBT. Since the light-induced oxidation of the Rieske center is only observed in the presence of antimycin, it seems likely that the reduced form of Q_z (Q_zH₂) reduces the Rieske center in an antimycin-sensitive reaction. From the extent of the UHDBT-sensitive ferricytochrome c₂ reduction we estimate that there are 0.7 Rieske iron-sulfur centers per reaction center.UHDBT shifts the EPR derivative absorption spectrum of the Rieske center from g_y 1.90 to g_y 1.89, and shifts the E_m,7 from 280 to 350 mV. While this latter shift may account for the subsequent failure of the iron-sulfur center to reduce ferricytochrome c₂, it is not clear how this can explain the other effects of the inhibitor, such as the prevention of cytochrome b reduction and the elimination of the uptake of H⁺_II; these may reflect additional sites of action of the inhibitor.     

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”.     

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.     

Formate dehydrogenase from Desulfovibrio desulfuricans ATCC 27774: isolation and spectroscopic characterization of the active sites (heme, iron-sulfur centers and molybdenum)

An air-stable formate dehydrogenase, an enzyme that catalyzes the oxidation of formate to CO₂, was purified from a sulfate-reducing organism, Desulfovibrio desulfuricans ATCC 27774. The enzyme has a molecular mass of approximately 150?kDa (three different subunits: 88, 29 and 16?kDa) and contains three types of redox-active centers: four c-type hemes, nonheme iron arranged as two [4Fe-4S]^2+/1+ centers and a molybdenum-pterin site. Selenium was also chemically detected. The enzyme specific activity is 78 units per mg of protein. Mo(V) EPR signals were observed in the native, reduced and formate-reacted states. EPR signals related to the presence of multiple low-spin hemes were also observed in the oxidized state. Upon reduction, an examination of the EPR data under appropriate conditions distinguishes two types of iron-sulfur centers, an [Fe-S] center I (g _max=2.050, g _med=1.947, g _min=1.896) and an [Fe-S] center II (g _max=2.071, g _med=1.926, g _min=1.865). Mössbauer spectroscopy confirmed the presence of four hemes in the low-spin state. The presence of two [4Fe-4S]^2+/1+ centers was confirmed, one of these displaying very small hyperfine coupling constants in the +1 oxidation state. The midpoint redox potentials of the enzyme metal centers were also estimated.     

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.     

Low temperature electron paramagnetic resonance studies on two iron-sulfur centers in cardiac succinate dehydrogenase

At temperatures below 20°K, EPR signals from a new iron-sulfur center (designated here as Center S-2 or (Fe-S)_S-2) in addition to the classical “g = 1.94 signal” (designated as Center S-1 or (Fe-S)_S-1) were detected in purified, soluble succinate dehydrogenase, particulate succinate ubiquinone reductase (Complex II) and particulate succinate cytochrome $\text{c}$ reductase from bovine heart. The measured half-reduction potential (E_m7.4) of Center S-1 was 0 ± 10 mV, while E_m7.4 of Center S-2 was ?260 ± 15 mV in the membrane bound preparations. Upon solubilization of succinate dehydrogenase, the EPR behavior of Center S-2 became extremely labile similar to the characteristics of the reconstitutive activity of succinate dehydrogenase toward the rest of the respiratory chain.     

Studies on electron paramagnetic resonance spectra manifested by a respiratory chain hydrogen carrier.

The high-potential iron-sulfur protein (HiPIP) center of succinate dehydrogenase has an electron paramagnetic resonance (epr) signal in the oxidized form, centered at g = 2.01, and under certain conditions this epr signal is accompanied by absorbances at g = 2.04, g = 1.99, and g = 1.96. These absorbances have been attributed to a spin-spin interaction of paramagnetic species, the semiquinone form of ubiquinone being involved (Ruzicka et al., Proc. Nat. Acad. Sci. USA72, 2886). In the present work this magnetic interaction is studied further; it is concluded that of the three possible species (HiPIP, Flavin H and UQ?H (ubiquinone)) which may interact with UQ?H; a second UQ? most likely partner for the interaction. Nonetheless, the HiPIP center of succinate dehydrogenase also plays a role in the interaction by acting as a “magnetic relaxer” of one or both of the interacting UQ?Hs. The physiological reaction of that part of the ubiquinone pool associated with the succinate dehydrogenase (on the matrix side of the inner mitochondrial membrane) is UQH₂ ? UQ?H + H⁺ + e^?. This is in line with recent postulates of the mechanism of ubiquinone mediation in electron transfer.     

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.     

Low-temperature electron transfer suggests two types of QA in intact photosystem II

The correlation between the reduction of Q_A and the oxidation of Tyr_Z or Car/Chl_Z/Cyt_b559 in spinach PSII enriched membranes induced by visible light at 10 K is studied by using electron paramagnetic resonance spectroscopy. Similar g = 1.95-1.86 Q_A^-•EPR signals are observed in both Mn-depleted and intact samples, and both signals are long lived at low temperatures. The presence of PPBQ significantly diminished the light induced EPR signals from Q_A^-•, Car^+•/Chl^+• and oxidized Cyt_b559, while enhancing the amplitude of the S₁Tyr_Z• EPR signal in the intact PSII sample. The quantification and stability of the g = 1.95-1.86 EPR signal and signals arising from the oxidized Tyr_Z and the side-path electron donors, respectively, indicate that the EPR-detectable g = 1.95-1.86 Q_A^-• signal is only correlated to reaction centers undergoing oxidation of the side-path electron donors (Car/Chl_Z/Cyt_b559), but not of Tyr_Z. These results imply that two types of Q_A^-• probably exist in the intact PSII sample. The structural difference and possible function of the two types of Q_A are discussed.     

Action of the antitumor and antispermatogenic agent lonidamine on electron transport in ehrlich ascites tumor mitochondria

The effect of lonidamine, an antispermatogenic and antitumor drug, on the oxygen consumption, ATPase activity, and redox state of the electron carriers of Ehrlich ascites tumor mitochondria has been studied. Lonidamine inhibits ADP- and uncoupler-stimulated respiration on various NAD- and FAD-linked substrates, but does not affect state 4 respiration. Experiments to determine its site of action showed that lonidamine does not significantly inhibit electron flow through cytochrome oxidase. Electron flow through site 2, the ubiquinone-cytochrome b-cytochrome c₁ complex, also was unaffected by lonidamine, which failed to inhibit the oxidation of duroquinol. Moreover, inhibition of electron flow through site 2 was also excluded because of the inability of the N,N,N′,N′-tetramethyl-p-phenylenediamine bypass to relieve the lonidamine inhibition of the oxidation of pyruvate + malate. The F₀F₁ATPase activity and vectorial H⁺ ejection are also unaffected by lonidamine. The inhibition of succinate oxidation by lonidamine was found to take place at a point between succinate and iron-sulfur center S3. Spectroscopic experiments demonstrated that lonidamine inhibits the reduction of mitochondrial NAD⁺ by pyruvate + malate and other NAD-linked substrates in the transition from state 1 to state 4. However, lonidamine does not inhibit reduction of added NAD⁺ by submitochondrial vesicles or by soluble purified NAD-linked dehydrogenases. These observations, together with other evidence, suggest that electron transport in tumor mitochondria is inhibited by lonidamine at the dehydrogenase-coenzyme level, particularly when the electron carriers are in a relatively oxidized state and/or when the inner membrane-matrix compartment is in the condensed state. The action of lonidamine in several respects resembles the selective inhibition of electron transport in tumor cells produced by cytotoxic macrophages.     

Regulation of succinate oxidation by NAD+ in mitochondria purified from potato tubers

NAD⁺ supplied to purified Solanum tuberosum mitochondria caused progressive inhibition of succinate oxidation in State 3. This inhibition was especially pronounced at alkaline pH and at low succinate concentrations. Glutamate counteracted the inhibition. NAD⁺ promoted oxaloacetate accumulation in State 3; supplied oxaloacetate inhibited O₂ uptake in the presence of succinate much more severely in State 3 than in State 4. NAD reduction linked to succinate oxidation by ATP-dependent reverse electron transport was likewise inhibited by oxaloacetate. We conclude that NAD⁺-induced inhibition of succinate oxidation is due to an inhibition of succinate dehydrogenase resulting from increased accumulation of oxaloacetate generated from malate oxidation via malate dehydrogenase. The results are discussed in the context of the known regulatory characteristics of plant succinate dehydrogenase.     

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).     

Electron transport and triplet formation in membranes of the photosynthetic bacterium Heliobacterium chlorum

The spectroscopic and thermodynamic properties of the electron-transport components of the photosynthetic bacterium Heliobacterium chlorum were studied by means of absorbance-difference spectroscopy. Upon flash illumination of membranes of H. chlorum photooxidation of the primary electron donor, P-798, was observed. In about 15% of the reaction centers P-798⁺ was reduced by cytochrome c-553, while in the remaining reaction centers P-798⁺ reduction occurred via a back reaction with a reduced electron acceptor. Titration experiments indicated a midpoint potential of −440 mV for the electron acceptor. At low redox potentials the formation of the triplet of P-798 was observed after a flash. The triplet was formed in about 30 ns by a back reaction with a reduced electron acceptor and decayed with a time constant of 35 μs. The yield of triplet formed in a flash was 30%. Upon continuous illumination at low redox potentials the accumulation in the reduced state of an electron acceptor was observed. The difference spectrum of this acceptor indicates that it is an iron-sulfur center. The yield of triplet formation was independent of the redox state of the iron-sulfur center, which indicates that the center is not located in the main electron-transport chain. A scheme with three acceptors in the main electron-transport chain is presented to accomodate our results and those of others.     

Studies on energy-linked reactions. Energy-linked reduction of oxidized nicotinamide–adenine dinucleotide by succinate in Escherichia coli

1. A method for the preparation of small particles from Escherichia coli is described. 2. These particles catalyse the ATP-driven reduction of NAD⁺ by succinate. 3. ATP utilized/NADH produced ratios below 2.0 were measured and a stoicheiometry of 1 molecule of ATP utilized per molecule of NADH produced is proposed. 4. The reaction is not inhibited by 2,4-dinitrophenol or by oligomycin but is inhibited by other uncouplers such as pentabromophenol and dicoumarol. 5. The specificity of the energy source, the specificity of the electron acceptor, the effects of pH, Mg²⁺, P_i and temperature have been studied.