Exchange integral for a variety of tetranuclear ferredoxins.

The temperature dependence of EPR spectra of oxidized [4Fe-4S](-1,-2) ferredoxins (previously designated HiPIP) and a reduced [4Fe-4S](-2,-3) ferredoxin have been analyzed so as to determine the energy of a low-lying excited electronic state. The values obtained were: Center S-3 from beef heart, 44 cm-1; Center S-3 from mung bean, 53 cm-1; the [4Fe-4S](-1,-2) ferredoxin from Thermus thermophilus, 78 cm-1; Center N-2 of NADH ubiquinone reductase, 83 cm-1. Increasing axial distortion in the EPR spectra of the [4Fe-4S](-1,-2), ferrodoxins was associated with higher energy differences. Center N-2, a [Fe-4S](-2,-3) iron-sulfur cluster does not fit this relationship.     

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).     

Effects of ethanol and acetaldehyde on iron-sulfure centers in the mitochondrial respiratory chain.

Incubation of submitochondrial particles with relatively low concentrations of ethanol (20–100 mm) or acetaldehyde (1–10 mm) produces alterations in the electron paramagnetic resonance spectra of the iron-sulfur centers in the NADH dehydrogenase segments of the respiratory chain. The iron-sulfur centers in the NADH dehydrogenase region are most sensitive to both ethanol and acetaldehyde, in comparison to the iron-sulfur centers in succinate dehydrogenase and the cytochrome b-c region. Centers N-3, 4, N-5, 6 and N-1b are affected after relatively short incubation periods (3–30 min) while center N-2 shows considerable sensitivity over somewhat longer incubations (20–90 min). The most ethanol-sensitive center in the succinate dehydrogenase region of the respiratory chain is high potential iron-sulfur protein-type center S-3. Potentiometric analysis shows that these alterations are not due to simple changes in the redox state caused by addition of dissolved oxygen. Changes in the electron paramagnetic resonance spectra can be correlated with decreased rates of oxidation of NADH and, to a lesser extent, succinate in both ethanol- and acetaldehyde-treated submitochondrial particles.     

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).     

Iron-sulfur N-1 clusters studied in NADH-ubiquinone oxidoreductase and in soluble NADH dehydrogenase

Two N-1 type iron-sulfur clusters in NADH-ubiquinone oxidoreductase (Complex I, EC 1.6.5.3) were potentiometrically resolved: one was titrated as a component with a midpoint oxidation-reduction potential of -335 mV at pH 8.0, and with an n-value equal to one; the other as an extremely low midpoint potential component (Em 8.0 less than -500 mV). These two clusters are tentatively assigned to N-1b and N-1a, respectively. Cluster N-1b is completely reducible with NADH and has a spin concentration of about 0.8/FMN. Its EPR spectrum can be simulated as a single rhombic component with principal g values of 2.019, 1.937, and 1.922, which correspond to the Center 1 reported earlier by Orme-Johnson, N. R., Hansen, R. E., and Beinert, H. (1974) J. Biol. Chem. 249, 1922-1927. At extremely low oxidation-reduction potentials (less than -450 mV), additional EPR signals emerge with apparent g values of gz = 2.03, gy = 1.95, and gx = 1.91, which we assign to cluster N-1a. It is difficult, however, to simulate the detailed spectral line shape of this component as a single rhombic component, suggesting some degree of protein modification or interaction with a neighboring oxidation-reduction component. EPR spectra of soluble NADH dehydrogenase, containing 5-6 g atoms of non-heme iron and 5-6 mol of acid-labile sulfide/mol of FMN, were examined. Signals from at least two iron-sulfur species could be distinguished in the NADH-reduced form: one of an N-1b type spectrum; the other of a spectrum with g values of 2.045, 1.95, and 1.87 (total of about 0.5 spin equivalents/FMN). This is the first example of an N-1 type signal detected in isolated soluble NADH dehydrogenase.     

Exchange integral for a variety of tetranuclear ferredoxins

The temperature dependence of EPR spectra of oxidized [4Fe-4S1]^{(?1, ?2)} ferredoxins (previously designated HiPIP) and a reduced [4Fe-4S1]^(?2,?3) ferredoxin have been analyzed so as to determine the energy of a low-lying excited electronic state. The values obtained were: Center S-3 from beef heart, 44 cm^?1; Center S-3 from mung bean, 53 cm^?1; the [4Fe-4S1]^(?1,?2) ferredoxin from Thermus thermophilus, 78 cm^?1; Center N-2 of NADH ubiquinone reductase, 83 cm^?1. Increasing axial distortion in the EPR spectra of the [4Fe-4S1]^(?1,?2) ferredoxins was associated with higher energy differences. Center N-2, a [4Fe-4S1]^(?2,?3) iron-sulfur cluster does not fit this relationship.     

Thermodynamic and EPR characteristics of a HiPIP-type iron-sulfur center in the succinate dehydrogenase of the respiratory chain.

In addition to the two species of ferredoxin-type iron-sulfur centers (Centers S-1 and S-2), a third iron-sulfur center (Center S-3), which is paramagnetic in the oxidezed state analogous to the bacterial high potential iron-sulfur protein, has bwen detected in the reconstitutively active soluble succinate dehydrogenase preparation. Midpoint potential (at pH 7.4) of Center S-3 determined in a particulate succinate-cytochrome c reductase is +60 +/- 15 mV. In soluble form, Center S-3 becomes extremely labile towards oxygen or ferricyanide plus phenazine methosulfate similar to reconstitutive activity of the dehydrogenase. Thus, even freshly prepared reconstitutively active enzyme preparations show EPR spectra of Center S-3 which correspond approximately to 0.5 eq per flavin; in particulate preparations this component was found in a 1:1 ratio to flavin. All reconstitutively inactive dehydrogenase preparations that Center S-3 is an innate constituent of succinate dehydrogenase and plays an important role in mediating electrons from the flavoprotein subunit to most probably ubiquinone and then to the cytochrome chain.     

Characterization by electron paramagnetic resonance and studies on subunit location and assembly of the iron-sulfur clusters of Bacillus subtilis succinate dehydrogenase

Succinate dehydrogenase is a conserved membrane-bound enzyme consisting of two nonidentical subunits: a flavo iron-sulfur protein (Fp) subunit, containing a covalently bound flavin, and an iron-sulfur protein (Ip) subunit. Bacillus subtilis succinate dehydrogenase in wild type bacteria and 12 well characterized succinate dehydrogenase-defective mutants were examined by low temperature EPR spectroscopy to characterize the enzyme and study subunit location and biosynthesis of its iron-sulfur clusters. The wild type B. subtilis enzyme contains iron-sulfur clusters which are analogous to clusters S-1 and S-3 of bovine heart succinate dehydrogenase but with slightly different EPR characteristics. Spins from cluster S-2 were not detectable as in the case of the intact form of bovine heart succinate dehydrogenase. However, dithionite reduction of the B. subtilis enzyme greatly enhanced spin relaxation of the ferredoxin-type cluster S-1, indicating the presence of the cluster S-2. Iron-sulfur cluster S-1 was found to be assembled in soluble succinate dehydrogenase subunits in the cytoplasm, but only if full-length Fp polypeptides and relatively large fragments of Ip polypeptides were present. Cluster S-1 was not detected in mutants with soluble mutated Fp polypeptides or in a mutant totally lacking Ip subunit polypeptide. Iron-sulfur clusters S-1, S-2, and S-3 were assembled also when the covalently bound flavin in the Fp subunit was absent. Clusters S-1 and S-3 in the membrane-bound flavin-deficient succinate dehydrogenase were not reduced by succinate but could be reduced by electron transfer from NADH dehydrogenase via the menaquinone pool.     

Identification of the iron-sulfur center of spinach ferredoxin-nitrite reductase as a tetranuclear center, and preliminary EPR studies of mechanism.

EPR spectroscopic and chemical analyses of spinach nitrite reductase show that the enzyme contains one reducible iron-sulfur center, and one site for binding either cyanide or nitrite, per siroheme. The heme is nearly all in the high spin ferric state in the enzyme as isolated. The extinction coefficient of the enzyme has been revised to E386 = 7.6 X 10(4) cm-1 (M heme)-1. The iron-sulfur center is reduced with difficulty by agents such as reduced methyl viologen (equilibrated with 1 atm of H2 at pH 7.7 in the presence of hydrogenase) or dithionite. Complexation of the enzyme with CO (a known ligand for nitrite reductase heme) markedly increases the reducibility of the iron-sulfur center. New chemical analyses and reinterpretation of previous data show that the enzyme contains 6 mol of iron and 4 mol of acid-labile S2-/mol of siroheme. The EPR spectrum of reduced nitrite reductase in 80% dimethyl sulfoxide establishes clearly that the enzyme contains a tetranuclear iron-sulfur (Fe4S4) center. The ferriheme and Fe4S4 centers are reduced at similar rates (k = 3 to 4 s-1) by dithionite. The dithionite-reduced Fe4S4 center is rapidly (k = 100 s-1) reoxidized by nitrite. These results indicate a role for the Fe4S4 center in catalysis.     

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.     

The spatial relationships and structure of the binuclear iron-sulfur clusters in succinate dehydrogenase.

Two binuclear iron-sulfur clusters (designated S-1 and S-2) are present in succinate dehydrogenase in approximately equal concentration to that of flavin. The large difference in their midpoint potentials (0 and -400 mV, respectively, in the soluble enzyme) permits the acquisition of individual electron paramagnetic resonance spectra characterized by nearly identical rhombic g tensors (gz = 2.025, gy = 1.93, gx = 1.905). Spin-coupling between the two centers is manifested by broadening and splitting of spectra of reconstitutively active and inactive succinate dehydrogenase, respectively, as the temperature is lowered; relief of power saturation of Center S-1 spectra on reduction of Center S 2; and observation of half-field ("delta ms = 2") signals in the dithionite-reduced enzyme. Saturation behavior of fully reduced dehydrogenase is consistent with the presence of S-1 and S-2 at equivalent concentrations/molecule. Simulation of the spin-coupled spectra, assuming dipolar interaction, provides information on molecular structure. Electron paramagnetic resonance spectra of the enzyme in 80% dimethylsulfoxide are nearly identical to the characteristic binuclear spectra obtained with adrenodoxin. These data provide additional evidence for binuclear structure of both Center S-1 and S-2. The extremely fast relaxation of Center S-2 at low temperatures would imply either an anomalously small value of J or an alternative relaxation mechanism, possibly due to the coupling between S-1 and S-2.     

Spectroscopic characterization of the number and type of iron-sulfur clusters in NADH:ubiquinone oxidoreductase

The number and type of iron-sulfur clusters present in the NADH dehydrogenase of the mammalian respiratory chain were studied by a combination of low temperature magnetic circular dichroism (MCD) and quantitative electron paramagnetic resonance spectroscopies. MCD was used with the high molecular weight, soluble enzyme, and EPR was used with both the purified enzyme and Complex I (NADH:ubiquinone oxidoreductase). The results of the EPR experiments of the two types of preparations agreed with each other, as well as with the data in the literature for various types of membrane-bound preparations. The two methods gave concordant results showing the presence of one binuclear and of three tetranuclear NADH-reducible iron-sulfur clusters. Earlier studies using the cluster extrusion technique indicated a higher ratio of binuclear to tetranuclear clusters which may be explained by cluster interconversion during the extrusion process.     

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.     

Function of conserved acidic residues in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica

Proton-translocating NADH:ubiquinone oxidoreductase (complex I) is the largest and least understood enzyme of the respiratory chain. Complex I from bovine mitochondria consists of more than forty different polypeptides. Subunit PSST has been suggested to carry iron-sulfur center N-2 and has more recently been shown to be involved in inhibitor binding. Due to its pH-dependent midpoint potential, N-2 has been proposed to play a central role both in ubiquinone reduction and proton pumping. To obtain more insight into the functional role of PSST, we have analyzed site-directed mutants of conserved acidic residues in the PSST homologous subunit of the obligate aerobic yeast Yarrowia lipolytica. Mutations D136N and E140Q provided functional evidence that conserved acidic residues in PSST play a central role in the proton translocating mechanism of complex I and also in the interaction with the substrate ubiquinone. When Glu(89), the residue that has been suggested to be the fourth ligand of iron-sulfur center N-2 was changed to glutamine, alanine, or cysteine, the EPR spectrum revealed an unchanged amount of this redox center but was shifted and broadened in the g(z) region. This indicates that Glu(89) is not a ligand of N-2. The results are discussedin the light of structural similarities to the homologous [NiFe] hydrogenases.     

Coupling site I and the rotenone-sensitive ubisemiquinone in tightly coupled submitochondrial particles

The rotenone-sensitive g = 2.00 low temperature EPR signal attributed to ubisemiquinone is observed in submitochondrial particles during coupled electron transfer from NADH to oxygen and from succinate to NAD+. The signal is seen only in the presence of oligomycin added to induce the respiratory control (7-9 with NADH and 3-4 with succinate) and it disappears in the presence of uncouplers (CCCP or gramicidin D). No reduction of the iron-sulfur center N-2 in the presence of 20 mM succinate and cyanide is observed, thus suggesting that N-2 is not in equilibrium with the ubiquinone pool. A hypothesis is proposed on delta mu H+ generation coupled with electron transfer between iron-sulfur center N-2 and the ubiquinone pool.     

Iron-sulfur centers in the Staphylococcus aureus respiratory chain]

Using low temperature EPR spectroscopy, signals of iron-sulfur centers with g-factors of 2.02 and 1.94 were detected in the respiratory chain of St. aureus membranes. According to their relaxation parameters and redox properties, these iron-sulfur centers are similar to iron-sulfur centers S-1 and S-3 corresponding to succinate dehydrogenases of mitochondria and bacterial membranes.     

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.     

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 X 10(-4) cm-1 and [E] = 39 X 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 delta Hpp = 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 delta Hpp = 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.     

Bacillus subtilis mutant succinate dehydrogenase lacking covalently bound flavin: identification of the primary defect and studies on the iron-sulfur clusters in mutated and wild-type enzyme

Succinate dehydrogenase consists of two protein subunits and contains one FAD and three iron-sulfur clusters. The flavin is covalently bound to a histidine in the larger, Fp, subunit. The reduction oxidation midpoint potentials of the clusters designated S-1, S-2, and S-3 in Bacillus subtilis wild-type membrane-bound enzyme were determined as +80, -240, and -25 mV, respectively. Magnetic spin interactions between clusters S-1 and S-2 and between S-1 and S-3 were detected by using EPR spectroscopy. The point mutations of four B. subtilis mutants with defective Fp subunits were mapped. The gene of the mutant specifically lacking covalently bound flavin in the enzyme was cloned. The mutation was determined from the DNA sequence as a glycine to aspartate substitution at a conserved site seven residues downstream from the histidine that binds the flavin in wild-type enzyme. The redox midpoint potential of the iron-sulfur clusters and the magnetic spin interactions in mutated succinate dehydrogenases were indistinguishable from the those of the wild type. This shows that flavin has no role in the measured magnetic spin interactions or in the structure and stability of the iron-sulfur clusters. It is concluded from sequence and mutant studies that conserved amino acid residues around the histidyl-FAD are important for FAD binding; however, amino acids located more than 100 residues downstream from the histidyl in the Fp subunit can also effect flavinylation.     

Spectroscopic studies on the iron-sulfur centers of milk xanthine oxidase

The optical electron paramagnetic resonance and M?ssbauer spectral properties of the two iron-sulfur centers present in milk xanthine oxidase have been reexamined. It is found in the case of the optical spectral change observed on reduction of the enzyme that the two centers contribute approximately equally, with a ratio of spectral contributions for Fe/S I and Fe/S II of 0.55:0.45. This conclusion is based both on the behavior of the spectral change at wavelengths where only the two iron-sulfur centers contribute to the spectral change (under experimental conditions minimizing the effect of flavin semiquinone) during reductive titrations and a comparison of the spectra of 1- and 2-electron reduced enzyme under different conditions. This very similar spectral weighting for the two centers applies throughout the visible region. In the case of the EPR spectra, it is found from computer simulation of the signals observed under nonsaturating conditions that iron-sulfur center II exhibits g values of 1.902, 1.991, and 2.110 and does not exhibit two g values above that for the free electron, as has been reported (Lowe, J., Lynden-Bell, R.M., and Bray, R. C. (1972) Biochem. J. 130, 239-249). The g values for iron-sulfur center I obtained from the simulations are 1.894, 1.932, and 2.022. Finally, M?ssbauer spectra of xanthine oxidase have been obtained, and it is found that while the two iron-sulfur centers are indistinguishable in the oxidized state, the ferrous iron in one of the reduced iron-sulfur centers exhibits an unusually large quadrupole coupling.