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.     

Studies on the NADH-menaquinone oxidoreductase segment of the respiratory chain in Thermus thermophilus HB-8

Five distinct low potential iron-sulfur clusters have been identified potentiometrically in the membrane particles from Thermus thermophilus HB-8. Three of these clusters (designated as [N-1H]T, [N-2H]T, and [N-3]T) exhibit the following midpoint redox potentials and g values (Em8.0 = -274 mV, gx,y,z = 1.93, 1.94, 2.02), (Em8.0 = -304 mV, gx,y,z = 1.89, 1.95, 2.04), and (Em8.0 = -289 mV, gx,y,z = 1.80, 1.83, 2.06), respectively. These clusters, one binuclear and two tetranuclear, have been shown to be components of the energy coupled NADH-menaquinone oxidoreductase complex (NADH dh I). They are reducible by NADH in the piericidin A-inhibited aerobic membrane particles as well as in the purified NADH dh I complex. Two additional very low potential iron-sulfur clusters (one binuclear, [N-1L]T, and one tetranuclear, [N-2L]T) were observed in membrane particles. These clusters possess the following physiochemical properties (Em8.0 = -418 mV, gx,y,z = 1.93, 19.5, 2.02) and (Em8.0 = -437 mV, gx,y,z = 1.89, 1.95, 2.04), respectively. No high potential tetranuclear cluster equivalent to the mitochondrial iron-sulfur cluster [N-2]B was found in this bacterial system. In membrane particles isolated from T. thermophilus HB-8 cells, four different semiquinone species have been identified based on their redox midpoint potentials [Em9(Q/QH2) = 40, -100, -160, -300 mV] and sensitivity to the quinone analogue inhibitor, 2-heptyl-4-hydroxy quinoline-N-oxide. Of these semiquinone species the -100 mV component has been suggested to be part of the NADH dehydrogenase. Piericidin A sensitive delta psi formation has been demonstrated to be coupled to the NADH-MQ1 oxidoreductase in membrane vesicles of T. thermophilus HB-8.     

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 degrees 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 titratins with EM7.2 values of minus 380 plus or minus 20 mV and minus 240 plus or minus 20 mV (Centers N-1a and N-1b, respectively). Center N-1a exhibits an EPR spectrum of nearly axial symmetry with g parellel = 2.03, g = 1.94, while that of Center N-1b shows more apparent rhombic symmetry with gz = 2.03, gy = 1.94 and gx = 1.91. Center N-2 also reveals EPR signals of axial symmetry at g parallel = 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 EM7.2 value (minus 20 plus or minus 20 mV). Resolution of Centers N-3 and N-4 was achieved potentiometrically in submitochondrial particles. The component with EM7.2 = minus 240 plus or minus 20 mV is defined as Center N-3 (gz = 2.10, (gz = 2.10, (gy = 1.93?), GX = 1.87); the minus 405 plus or minus 20 mV component as Center N-4 (gz = 2.11, (gy = 1.93?), gx = 1.88). At temperatures close to 4.2 degrees K, EPR signals at g = 2.11, 2.06, 2.03, 1.93, 1.90 and 1.88 titrate with EM7.2 = minus 260 plus or minus 20 mV. The multiplicity of peaks suggests the presence of at least two different iron-sulfur centers having similar EM7.2 values (minus 260 plus or minus 20 mV); HENCE, tentatively assigned as N-5 and N-6. Consistent with the individual EM7.2 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 EM7.2 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. EM7.2 values and EPR lineshapes for the other iron-sulfur 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 EM7.2 values around minus 250 mV; Center N-2 exhibits a high EM7.2 (minus 20 plus or minus 20 mV), while Center N-1a shows a very low EM7.2 (minus 380 plus or minus 20 mV).     

EPR characterization of the iron-sulfur clusters in the NADH: ubiquinone oxidoreductase segment of the respiratory chain in Paracoccus denitrificans

The physicochemical properties of the iron-sulfur clusters present in the NADH:ubiquinone oxidoreductase of Paracoccus denitrificans have been examined in the cytoplasmic membrane particles by redox potentiometry and EPR spectroscopy. Analogous to the iron-sulfur clusters present in the mitochondrial NADH: ubiquinone oxidoreductase, we have found two binuclear and three tetranuclear EPR detectable iron-sulfur clusters, namely, N-1a, N-1b, N-2, N-3, and N-4. In the bacterial system, the two binuclear clusters differ in line shape and in Em values; the cluster with more rhombic symmetry (gx,y,z = 1.918, 1.937, 2.029) has the Em7.0 value of -150 while the almost axial one (gx,y,z = 1.929, 1.941, 2.019) has Em7.0 of -270 mV. The Em of the former cluster is pH dependent (-60 mV/pH) as in the case of mammalian N-1a while the latter is pH independent as is the mammalian cluster N-1b. The pH-dependent P. denitrificans [2Fe-2S] cluster, which we have labeled N-1a, has an Em7.0 as high as that of N-2, in contrast to the mammalian N-1a. Thus N-1a is reducible with a physiological reductant, NADH in this bacterial system. The Em of the cluster N-2 is also pH dependent (Em7.0 = -130 mV) with a pK value near 7.7. The Em values of all other clusters exhibit no pH dependence as in the case of their mammalian counterparts. We have found that the cluster N-1a is the most labile component among the five iron-sulfur clusters and may give rise to variable relative spin concentrations and extremely low Em values due to the facile modifications of the microenvironment of the cluster. The P. denitrificans NADH:ubiquinone oxidoreductase provides a unique and useful site I model system where redox composition is similar to the mitochondrial enzyme but with fewer numbers of polypeptides (Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311).     

Determination of the exchange integral in binuclear iron-sulfur clusters in proteins of varying complexity.

The coupling constants J between the iron atoms in ferredoxin type iron-sulfur proteins containing binuclear clusters were evaluated by two parallel methods. The temperature dependence of the EPR linewidths and integrated abosrption intensities are both related to the energy of the first excited state. The values of J obtained were: center S-1 in succinate dehydrogenase, 90 cm-1; Rieske's iron-sulfur center, 65 cm-1; adrenodoxin, 270 cm-1. The behavior of iron-sulfur center N-1a in NADH:UQ reductase was also examined; its similarity to that of center S-1 indicates that center N-1a is also a binuclear iron-sulfur center, with J = 90 cm-1. Greater rhombic distortion present in the EPR spectrum of a binuclear cluster was associated with smaller values of J.     

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.     

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.     

EPR characterization of the iron-sulfur-containing NADH-ubiquinone oxidoreductase of the Escherichia coli aerobic respiratory chain

The energy coupled NADH-ubiquinone (Q) oxidoreductase segment of the respiratory chain of Escherichia coli GR19N has been studied by EPR spectroscopy. Previously Matsushita et al. [(1987) Biochemistry 26, 7732-7737] have demonstrated the presence of two distinct NADH-Q oxidoreductases in E. coli membrane particles and designated them NADH dh I and NADH dh II. Although both enzymes oxidize NADH, only NADH dh I is coupled to the formation of the H+ electrochemical gradient. In addition to NADH, NADH dh I oxidizes nicotinamide hypoxanthine dinucleotide (deamino-NADH), while NADH dh II does not. In membrane particles we have detected EPR signals arising from four low-potential iron-sulfur clusters, one binuclear, one tetranuclear, and two fast spin relaxing g perpendicular = 1.94 type clusters (whose cluster structure has not yet been assigned). The binuclear cluster, temporarily designated [N-1]E, shows an EPR spectrum with gx,y,z = 1.92, 1.935, 2.03 and the Em7.4 value of -220 mV (n = 1). The tetranuclear cluster, [N-2]E, elicits a spectrum with gx,y,z = 1.90, 1.91, 2.05 and an Em7.4 of -240 mV (n = 1). These two clusters have been shown to be part of the NADH dh I complex by stability and inhibitor studies. When stored at 4 degrees C, both clusters are extremely labile as is the deamino-NADH-Q oxidoreductase activity. Addition of deamino-NADH in the presence of piericidin A results in nearly full reduction of [N-2]E within 17 s. In membrane particles pretreated with piericidin A, the cluster [N-1]E is only partly reducible by deamino-NADH and shows an altered line shape.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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).     

Iron-sulphur centres in mitochondria from Arum maculatum spadix with very high rates of cyanide-resistant respiration.

X-band electron-paramagnetic-resonance spectroscopy at 4.2--77K combined with measurements of oxidation-reduction potential was used to identify iron--sulphur centres in Arum maculatum (cuckoo-pint) mitochondria. In the oxidized state a signal with a derivative maximum at g = 2.02 was assigned to succinate dehydrogenase centre S-3. Unreduced particles showed additional signals at g = 2.04 and 1.98 (at 9.2 GHz), which may be due to a spin-spin interaction. In the reduced state a prominent signal at g = 1.93 and 2.02 was resolved into at least three components that could be assigned to centres S-1 and S-2 of succinate dehydrogenase (midpoint potentials -7 and -240 mV respectively at pH 7.2) and a small amount of centre N-1b (e'o= -240 mV) of NADH-ubiquinone reductase. In addition, changes in line shape around -10 mV indicated the presence of a fourth component in this signal. The latter was more readily reduced by NADH than by succinate, suggesting that it might be associated with the external NADH dehydrogenase. The iron-sulphur centres of NADH-ubiquinone reductase were present in an unusually low concentration, indicating that the alternative, non-phosphorylating, NADH dehydrogenase containing a low number of iron-sulphur centres may be responsible for most of the high rate of oxidation of NADH.     

The Fe-only nitrogenase and the Mo nitrogenase from Rhodobacter capsulatus: a comparative study on the redox properties of the metal clusters present in the dinitrogenase components.

The dinitrogenase component proteins of the conventional Mo nitrogenase (MoFe protein) and of the alternative Fe-only nitrogenase (FeFe protein) were both isolated and purified from Rhodobacter capsulatus, redox-titrated according to the same procedures and subjected to an EPR spectroscopic comparison. In the course of an oxidative titration of the MoFe protein (Rc1Mo) three significant S = 1/2 EPR signals deriving from oxidized states of the P-cluster were detected: (1) a rhombic signal (g = 2.07, 1.96 and 1.83), which showed a bell-shaped redox curve with midpoint potentials (Em) of -195 mV (appearance) and -30 mV (disappearance), (2) an axial signal (g(parallel) = 2.00, g perpendicular = 1.90) with almost identical redox properties and (3) a second rhombic signal (g = 2.03, 2.00, 1.90) at higher redox potentials (> 100 mV). While the 'low-potential' rhombic signal and the axial signal have been both attributed to the one-electron-oxidized P-cluster (P1+) present in two conformationally different proteins, the 'high-potential' rhombic signal has been suggested rather to derive from the P3+ state. Upon oxidation, the FeFe protein (Rc1Fe) exhibited three significant S = 1/2 EPR signals as well. However, the Rc1Fe signals strongly deviated from the MoFe protein signals, suggesting that they cannot simply be assigned to different P-cluster states. (a) The most prominent feature is an unusually broad signal at g = 2.27 and 2.06, which proved to be fully reversible and to correlate with catalytic activity. The cluster giving rise to this signal appears to be involved in the transfer of two electrons. The midpoint potentials determined were: -80 mV (appearance) and 70 mV (disappearance). (b) Under weakly acidic conditions (pH 6.4) a slightly altered EPR signal occurred. It was characterized by a shift of the g values to 2.22 and 2.05 and by the appearance of an additional negative absorption-shaped peak at g = 1.86. (c) A very narrow rhombic EPR signal at g = 2.00, 1.98 and 1.96 appeared at positive redox potentials (Em = 80 mV, intensity maximum at 160 mV). Another novel S = 1/2 signal at g = 1.96, 1.92 and 1.77 was observed on further, enzymatic reduction of the dithionite-reduced state of Rc1Fe with the dinitrogenase reductase component (Rc2Fe) of the same enzyme system (turnover conditions in the presence of N2 and ATP). When the Rc1Mo protein was treated analogously, neither this 'turnover signal' nor any other S = 1/2 signal were detectable. All Rc1Fe-specific EPR signals detected are discussed and tentatively assigned with special consideration of the reference spectra obtained from Rc1Mo preparations.     

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.     

A 4-methoxybenzoate O-demethylase from Pseudomonas putida. A new type of monooxygenase system.

A strain of Pseudomonas putida grown on 4-methoxybenzoate as sole carbon source contains an enzyme system for the O-demethylation of this substrate. The enzyme system is purifiable and can be separated into two components: an NADH-dependent reductase and an iron-containing and acid-labile-sulfur-containing monooxygenase. The reductase, of molecular weight 42000 and containing two chromophores, an FMN and an iron-sulfur complex (EPR at g = 1.95), reduces both one-electron and two-electron acceptors (i.e., ferricyanide, 2,6-dichloroindophenol, cytochrome c, and cytochrome b5) at an optimum pH of 8.0. Increasing ionic strength affects these activities differently. The absolute spectrum of the oxidized displays distinct absorption peaks at 409 and 463 nm and a small shoulder between 538 and 554 nm. Treatment with dithionite or NADH reduces the absorbance throughout the visible range, yielding a spectrum with small maxima at 402 and 538 nm. Spectroscopic characteristics of the reductase indicate a tight coupling between its two chromophores. The iron-containing and acid-labile-sulfur-containing monooxygenase, which has a molecular weight of about 120000, contains an iron-sulfur chromophore with an EPR signal at g = 1.90. This protein is a dimer whose subunits each have a molecular weight of about 50000 and are perhaps identical. The optical absorption properties are somewhat unusual. In contrast to other iron-sulfur proteins, there is no significant peak near 415 nm in the absorption spectrum of the oxidized protein, but rather one at 455 nm. The presence of the substrate 4-methoxybenzoate increases both the NADH-dependent reductase. Hydroxylation can be achieved by the monooxygenase also in absence of the reductase with artifical reductants. This enzyme opens a new group of oxygenases within the classification scheme, i.e., iron-containing and labile-sulfur-containing monooxygenases. From the reported data, a scheme for the interaction of the isolated pigments and their relationship to various acceptors is proposed.     

Electrostatic interactions between FeS clusters in NADH:ubiquinone oxidoreductase (Complex I) from Escherichia coli

The redox properties of the cofactors of NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli were studied by following the changes in electron paramagnetic resonance (EPR) and optical spectra upon electrochemical redox titration of the purified protein. At neutral pH, the FMN cofactor had a midpoint redox potential ( E m) approximately -350 mV ( n = 2). Binuclear FeS clusters were well-characterized: N1a was titrated with a single ( n = 1) transition, and E m = -235 mV. In contrast, the titration of N1b can only be fitted with the sum of at least two one-electron Nernstian curves with E m values of -245 and -320 mV. The tetranuclear clusters can also be separated into two groups, either having a single, n = 1, or more complex redox titration curves. The titration curves of the EPR bands attributed to the tetranuclear clusters N2 ( g = 2.045 and g = 1.895) and N6b ( g = 2.089 and g = 1.877) can be presented by the sum of at least two components, each with E m (app) approximately -200/-300 mV and -235/-315 mV, respectively. The titration of the signals at g = 1.956-1.947 (N3 or N7, E m = -315 mV), g = 2.022, and g = 1.932 (Nx, -365 mV) and the low temperature signal at g = 1.929 (N4 or N5, -330 mV) followed Nernstian n = 1 curves. The observed redox titration curves are discussed in terms of intrinsic electrostatic interactions between FeS centers in complex I. A model showing shifts of E m due to the electrostatic interaction between the centers is presented.     

The preparation and characterization of highly purified, enzymically active complex III from baker's yeast.

A soluble enzymically active cytochrome b.c1 complex has been purified from baker's yeast mitochondria by a procedure involving solubilization in cholate, differential fractionation with ammonium sulfate, and ultracentrifugation. The resulting particle is free of both cytochrome c oxidase and succinate dehydrogenase activities. The complex contains cytochromes b and c1 in a ratio of 2:1 and quinone and iron-sulfur protein in amounts roughly stoichiometric with cytochrome c1. EPR spectroscopy has shown the iron-sulfur protein to be present mainly as the Rieske protein. EPR spectroscopy also shows a heterogeneity in the cytochrome b population with resonances appearing at g = 3.60 (cytochrome bK) and g = 3.76 (cytochrome bT). A third EPR resonance appearing in the region associated with low spin ferric hemes (g = 3.49) is assigned to cytochrome c1. Anaerobic titration of the complex with dithionite confirmed the heterogeneity in the cytochrome b population and demonstrated that the oxidation-reduction potential of the iron-sulfur protein is approximately 30 mV more positive than cytochrome c1. An intense EPR signal assigned to the coenzyme Q free radical appeared midway in the reductive titration; this signal disappeared toward the end of the titration. A conformational change in the iron-sulfur protein attendant on reduction of a low potential species was noted.     

The NAD-linked soluble hydrogenase from Alcaligenes eutrophus H16: detection and characterization of EPR signals deriving from nickel and flavin

 In this study we confirmed the previous observation that the cytoplasmic NAD-linked hydrogenase of Alcaligenes eutrophus H16 is EPR-silent in the oxidized state. We also demonstrated the presence of significant Ni-EPR signals when the enzyme was either reduced with the natural electron carrier NADH (5–10 mM) or carefully titrated with sodium dithionite to an intermediate, narrow redox potential range (–280 to –350 mV). Reduction with NADH under argon atmosphere led to a complex EPR spectrum at 80 K with g values at 2.28, 2.20, 2.14, 2.10, 2.05, 2.01 and 2.00. This spectrum could be differentiated by special light/dark treatments into three distinct signals: (1) the "classical" Ni-C signal with g values at 2.20, 2.14 and 2.01, observed with many hydrogenases in the reduced, active state; (2) the light-induced signal (Ni-L) with g values at 2.28, 2.10 and 2.05 and (3) a flavin radical (FMN semiquinone) signal at g = 2.00. The assignment of the Ni-EPR signal was clearly confirmed by EPR spectra of hydrogenase labeled with ⁶¹Ni (nuclear spin I = 3/2) yielding a broadening of the Ni spectra at all g values and a resolved ⁶¹Ni hyperfine splitting into four lines of the low field edge in the case of the light-induced Ni-EPR signal. The redox potentials determined at pH 7.0 for the described redox components were: for FMN –170 mV (midpoint potential, E_m, for appearance), –200 mV (EPR signal intensity maximum) and –230 mV (E_m for disappearance); for the Ni centre (Ni-C), –290 mV (E_m for appearance), –305 mV (signal intensity maximum) and –325 mV (E_m for disappearance). Exposure of the NADH-reduced hydrogenase to carbon monoxide led to an apparent Ni-CO species indicated by a novel rhombic EPR signal with g values at 2.35, 2.08 and 2.01. Received: 19 July 1995 / Accepted: 10 September 1995     

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.     

Purification, characterization and redox properties of hydrogenase from Methanosarcina barkeri (DSM 800)

A soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri (DSM 800) has been purified to apparent electrophoretic homogeneity, with an overall 550-fold purification, a 45% yield and a final specific activity of 270 mumol H2 evolved min-1 (mg protein)-1. The hydrogenase has a high molecular mass of approximately equal to 800 kDa and subunits with molecular masses of approximately equal to 60 kDa. The enzyme is stable to heating at 65 degrees C and to exposure to air at 4 degrees C in the oxidized state for periods up to a week. The overall stability of this enzyme is compared with other hydrogenase isolated from strict anaerobic sulfate-reducing bacteria. Ms. barkeri hydrogenase shows an absorption spectrum typical of a non-heme iron protein with maxima at 275 nm, 380 nm and 405 nm. A flavin component, identified as FMN or riboflavin was extracted under acidic conditions and quantified to approximately one flavin molecule per subunit. In addition to this component, 8-10 iron atoms and 0.6-0.8 nickel atom were also detected per subunit. The electron paramagnetic resonance (EPR) spectrum of the native enzyme shows a rhombic signal with g values at 2.24, 2.20 and approximately equal to 2.0. probably due to nickel which is optimally measured at 40 K but still detectable at 77 K. In the reduced state, using dithionite or molecular hydrogen as reductants, at least two types of g = 1.94 EPR signals, due to iron-sulfur centers, could be detected and differentiated on the basis of power and temperature dependence. Center I has g values at 2.04, 1.90 and 1.86, while center II has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite the rhombic EPR species disappears and is replaced by other EPR-active species with g values at 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only observable at temperatures higher than 20 K. In the native preparation, at high temperatures (T greater than 35 K) or in partially reduced samples, a free radical due to the flavin moiety is observed. The EPR spectrum of reduced hydrogenase in 80% Me2SO presents an axial type of spectrum only detectable below 30 K.     

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.