Isolation and characterization of rubrerythrin, a non-heme iron protein from Desulfovibrio vulgaris that contains rubredoxin centers and a hemerythrin-like binuclear iron cluster

A new non-heme iron protein from the periplasmic fraction of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) has been purified to homogeneity, and its amino acid composition, molecular weight, redox potential, iron content, and optical, EPR, and M?ssbauer spectroscopic properties have been determined. This new protein is composed of two identical subunits with subunit molecular weight of 21,900 and contains four iron atoms per molecule. The as-purified oxidized protein exhibits an optical spectrum with absorption maxima at 492, 365, and 280 nm, and its EPR spectrum shows resonances at g = 4.3 and 9.4, characteristic of oxidized rubredoxin. The M?ssbauer data indicate the presence of approximately equal amounts of two types of iron; we named them the Rd-like and the Hr-like iron due to their similarity to the iron centers of rubredoxins (Rds) and hemerythrins (Hrs), respectively. For the Rd-like iron, the measured fine and hyperfine parameters (D = 1.5 cm-1, E/D = 0.26, delta EQ = -0.55 mm/s, delta = 0.27 mm/s, Axx/gn beta n = -16.5 T, Ayy/gn beta n = -15.6 T, and Azz/gn beta n = -17.0 T) are almost identical with those obtained for the rubredoxin from Clostridium pasteurianum. Redox-titration studies monitored by EPR, however, showed that these Rd-like centers have a midpoint redox potential of +230 +/- 10 mV, approximately 250 mV more positive than those reported for rubredoxins. Another unusual feature of this protein is the presence of the Hr-like iron atoms.(ABSTRACT TRUNCATED AT 250 WORDS)     

The iron-sulphur centres of soluble hydrogenase from Alcaligenes eutrophus.

The soluble hydrogenase (hydrogen:NAD+ oxidoreductase (EC 1.12.1.2) from Alcaligenes eutrophus has been purified to homogeneity by an improved procedure, which includes preparative electrophoresis as final step. The specific activity of 57 mumol H2 oxidized/min per mg protein was achieved and the yield of pure enzyme from 200 g cells (wet weight) was about 16 mg/purification. After removal of non-functional iron, analysis of iron and acid-labile sulphur yielded average values of 11.5 and 12.9 atoms/molecule of enzyme, respectively. p-Chloromercuribenzoate was a strong inhibitor of hydrogenase and apparently competed with NAD not with H2. Chelating agents, CO and O2 failed to inhibit enzyme activity. The oxidized hydrogenase showed an EPR spectrum with a small signal at g = 2.02. On reduction the appearance of a high temperature (50--77 K) signal at g = 2.04, 1.95 and a more complex low temperature (less than 30 K) spectrum at g = 2.04, 2.0, 1.95, 1.93, 1.86 was observed. The pronounced temperature dependence and characteristic lineshape of the signals obtained with hydrogenase in 80--85% dimethylsulphoxide demonstrated that iron-sulphur centres of both the [2Fe-2S] and [4Fe-4S] types are present in the enzyme. Quantitation of the EPR signals indicated the existence of two identical centres each of the [4Fe-4S] and of the [2Fe-2S] type. The midpoint redox potentials of the [4Fe-4S] and the [2Fe-2S] centres were determined to be -445 mV and -325 mV, respectively. Spin coupling between two centres, indicated by the split feature of the low temperature spectrum of the native hydrogenase around g = 1.95, 1.93, has been established by power saturation studies. On reduction of the [Fe-4S] centres, the electron spin relaxation rate of the [2Fe-2S] centres was considerably increased. Treatment of hydrogenase with CO caused no change in EPR spectra.     

Purification and characterization of desulfoferrodoxin. A novel protein from Desulfovibrio desulfuricans (ATCC 27774) and from Desulfovibrio vulgaris (strain Hildenborough) that contains a distorted rubredoxin center and a mononuclear ferrous center

A new type of non-heme iron protein was purified to homogeneity from extracts of Desulfovibrio desulfuricans (ATCC 27774) and Desulfovibrio vulgaris (strain Hildenborough). This protein is a monomer of 16-kDa containing two iron atoms per molecule. The visible spectrum has maxima at 495, 368, and 279 nm and the EPR spectrum of the native form shows resonances at g = 7.7, 5.7, 4.1 and 1.8 characteristic of a high-spin ferric ion (S = 5/2) with E/D = 0.08. M?ssbauer data indicates the presence of two types of iron: an FeS4 site very similar to that found in desulforedoxin from Desulfovibrio gigas and an octahedral coordinated high-spin ferrous site most probably with nitrogen/oxygen-containing ligands. Due to this rather unusual combination of active centers, this novel protein is named desulfoferrodoxin. Based on NH2-terminal amino acid sequence determined so far, the desulfoferrodoxin isolated from D. desulfuricans (ATCC 27774) appears to be a close analogue to a recently discovered gene product from D. vulgaris (Brumlik, M.J., and Voordouw, G. (1989) J. Bacteriol. 171, 49996-50004), which was suggested to be a rubredoxin oxidoreductase. However, reduced pyridine nucleotides failed to reduce the desulforedoxin-like center of this new protein.     

Characterization of the soluble hydrogenase from Desulfovibrio africanus

The soluble hydrogenase from Desulfovibrio africanus has been isolated and characterized. The enzyme consists of two subunits of 65 kDa and 27 kDa. Its absorption spectrum is typical of an iron-sulfur protein. The protein contains 12 iron atoms, 10 labile sulfur atoms and 0.9 nickel atom per molecule. D. africanus hydrogenase is rapidly activated under reducing conditions and exhibits a specific activity of 570 mumoles H2 evolved/min/mg. The EPR spectrum of the oxidized enzyme shows no Ni(III) signals. Upon reduction under hydrogen, the protein sample exhibits signals due to nickel with g values at 2.21, 2.17 and 2.01 correlating with the active state of the enzyme.     

M?ssbauer and EPR spectroscopy of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) from Pseudomonas aeruginosa has been investigated by EPR and M?ssbauer spectroscopy. Low temperature M?ssbauer data on the native enzyme (Fe3+, S = 5/2) yields a hyperfine field Hsat=-525 kG at the nucleus. This observation is inconsistent with earlier suggestions, based on EPR data of a rubredoxin-like ligand environment around the iron, i.e. a tetrahedral sulfur coordination. Likewise, the dithionite-reduced enzyme has M?ssbauer parameters unlike those of reduced rubredoxin. We conclude that the iron atoms are in a previously unrecognized environment. The ternary complex of the enzyme with 3,4-dihydroxyphenylpropionate and O2 yields EPR signals at g = 6.7 and g = 5.3; these signals result from an excited state Kramers doublet. The kinetics of the disappearance of these signals parallels product formation and the decay of the ternary complex as observed in the optical spectrum. The M?ssbauer and EPR data on the ternary complex establish the iron atoms to be a high-spin ferric state characterized by a large and negative zero-field splitting, D = approximately -2 cm-1.     

Low-temperature magnetic circular dichroism spectra and magnetisation curves of 4Fe clusters in iron-sulphur proteins from Chromatium and Clostridium pasteurianum

The magnetic circular dichroism (MCD) spectra of the 4Fe clusters in the iron-sulphur proteins high-potential iron protein from Chromatium and the 8Fe ferredoxin from Clostridium pasteurianum have been measured over the wavelength range 300-800 nm at temperatures between approx. 1.5 and 50 K and at magnetic fields up to 5 tesla. In both cases the proteins have been studied in the oxidized and reduced states. The reduced state of high-potential iron protein gives a temperature-independent MCD spectrum up to 20 K, confirming the diamagetism of this state at low temperature. The MCD spectrum of samples of oxidized ferredoxin invariably show the presence of a low concentration of a paramagnetic species, in agreement with the observation that the EPR spectrum always shows a signal at g = 2.01. The paramagnetic MCD spectrum runs across the whole of the wavelength range studied and therefore most probably originates from an iron-sulphur centre. The diamagnetic component of the MCD spectrum of oxidized ferredoxin is very similar to that of reduced high-potential iron protein. The low-temperature MCD spectra of oxidized high-potential iron protein and reduced ferredoxin reveal intense, temperature-dependent bands. The spectra are highly structured with that of high-potential iron protein showing a large number of electronic transitions across the visible region. The MCD spectra of the two different oxidation levels are quite distinctive and should provide a means of establishing the identity of these state of 4Fe clusters in more complex proteins. MCD magnetisation curves have been constructed from detailed studies of the field and temperature dependence of the MCD spectra of the two paramagnetic oxidation states. These plots can be satisfactorily fitted to the theoretically computed curves for an S = 1/2 ground state with the g factors experimentally determined by EPR spectroscopy. The low-temperature MCD spectra of the reduced 2Fe-2S ferredoxin from Spirulina maxima are also presented and MCD magnetisation curves plotted and fitted to the experimentally determined g factors.     

Purification and properties of the membrane-bound hydrogenase from Proteus mirabilis.

The cytoplasmic membrane-bound hydrogenase of the facultative anaerobe, Proteus mirabilis, has been solubilized and purified to homogeneity. The purified enzyme exhibited a maximal specific activity of about 780 mumol H2 oxidized/min per mg protein (benzyl viologen reduction). The hydrogenase has a molecular weight of 205 000 and is composed of two subunits with a molecular weight of 63 000 and two of 33 000. The absorption spectrum of the enzyme was characteristic of non-heme iron proteins. The millimolar extinction coefficients at 400 and 280 nm are 106 and 390, respectively. The hydrogenase has about 24 iron atoms and 24 acid-labile sulfide atoms/molecule. Amino acid analyses revealed the presence of 39 half-cystine residues/molecule and a preponderance of acidic amino acids. The hydrogenase in its oxidized form exhibits an EPR signal of the HiPIP-type with g values at 2.025 and 2.018. Upon reduction with either dithionite or H2 the signal disappears; no other signals were detectable.     

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.     

The three-iron cluster in a ferredoxin from Desulphovibrio gigas. A low-temperature magnetic circular dichroism study

Ferredoxin II from Desulphovibrio gigas is a tetrameric protein containing a novel iron-sulphur cluster consisting of three iron atoms. The low-temperature magnetic circular dichroism (MCD) spectra of the oxidized and dithionite-reduced forms of ferredoxin II have been measured over the wavelength range approx. 300-800 nm. Both oxidation levels of the cluster are shown to be paramagnetic, although only the oxidized form gives an EPR signal. MCD magnetization curves have been constructed over the temperature range approx. 1.5-150 K and at fields between 0 and 5.1 Tesla. The curve for the oxidized protein can be fitted to a ground state of spin S = 1/2 with an isotropic g factor of 2.01. There is evidence for the thermal population of a low-lying electronic state above 50 K. The reduced protein gives a distinctive set of magnetization curves that are tentatively assigned to a ground state of S = 2, with a predominantly axial zero-field distortion that leaves the doublet Ms = +/-2 lowest in energy. The zero-field components have a maximum energy spread of approx. 15 cm-1. which places an upper limit of 4 cm-1 on the axial zero-field parameter D. The MCD spectra of the oxidized and reduced forms of the cluster are quite distinctive from one another. The spectra of the oxidized state are also different from those of oxidized high-potential iron protein from Chromatium and should provide a useful criterion for distinguishing between four- and three-iron clusters in their highest oxidation levels.     

Direct spectroscopic evidence for the presence of a 6Fe cluster in an iron-sulfur protein isolated from Desulfovibrio desulfuricans (ATCC 27774)

A novel iron-sulfur protein was purified from the extract of Desulfovibrio desulfuricans (ATCC 27774) to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a monomer of 57 kDa molecular mass. It contains comparable amounts of iron and inorganic labile sulfur atoms and exhibits an optical spectrum typical of iron-sulfur proteins with maxima at 400, 305, and 280 nm. M?ssbauer data of the as-isolated protein show two spectral components, a paramagnetic and a diamagnetic, of equal intensity. Detailed analysis of the paramagnetic component reveals six distinct antiferromagnetically coupled iron sites, providing direct spectroscopic evidence for the presence of a 6Fe cluster in this newly purified protein. One of the iron sites exhibits parameters (delta EQ = 2.67 +/- 0.03 mm/s and delta = 1.09 +/- 0.02 mm/s at 140 K) typical for high spin ferrous ion; the observed large isomer shift indicates an iron environment that is distinct from the tetrahedral sulfur coordination commonly observed for the iron atoms in iron-sulfur clusters and is consistent with a penta- or hexacoordination containing N and/or O ligands. The other five iron sites are most probably high spin ferric. Three of them show parameters characteristic for tetrahedral sulfur coordination. In correlation with the EPR spectrum of the as-purified protein which shows a resonance signal at g = 15.3 and a group of signals between g = 9.8 and 5.4, this 6Fe cluster is assigned to an unusual spin state of 9/2 with zero field splitting parameters D = -1.3 cm-1 and E/D = 0.062. Other EPR signals attributable to minor impurities are also observed at the g = 4.3 and 2.0 regions. The diamagnetic M?ssbauer component represents a second iron cluster, which, upon reduction with dithionite, displays an intense S = 1/2 EPR signal with g values at 2.00, 1.83, and 1.31. In addition, an EPR signal of the S = 3/2 type is also observed for the dithionite-reduced protein.     

Electron paramagnetic resonance studies of Pseudomonas cytochrome c peroxidase

The EPR spectrum at 15 K of Pseudomonas cytochrome c peroxidase, which contains two hemes per molecule, is in the totally ferric form characteristic of low-spin heme giving two sets of g-values with gz 3.26 and 2.94. These values indicate an imidazole-nitrogen : heme-iron : methionine-sulfur and an imidazole-nitrogen : heme-iron : imidazole-nitrogen hemochrome structure, respectively. The spectrum is essentially identical at pH 6.0 and 4.6 and shows only a very small amount of high-spin heme iron (g 5--6) also at 77 K. Interaction between the two hemes is shown to exist by experiments in which one heme is reduced. This induces a change of the EPR signal of the other (to gz 2.83, gy 2.35 and gx 1.54), indicative of the removal of a histidine proton from that heme, which is axially coordinated to two histidine residues. If hydrogen peroxide is added to the partially reduced protein, its EPR signal is replaced by still other signals (gz 3.5 and 3.15). Only a very small free radical peak could be observed consistent with earlier mechanistic proposals. Contrary to the EPR spectra recorded at low temperature, the optical absorption spectra of both totally oxidized and partially reduced enzyme reveal the presence of high-spin heme at room temperature. It seems that a transition of one of the heme c moieties from an essentially high-spin to a low-spin form takes place on cooling the enzyme from 298 to 15 K.     

A bacterioferritin from the strict anaerobe Desulfovibrio desulfuricans ATCC 27774

A bacterioferritin was isolated from the anaerobic bacterium Desulfovibrio desulfuricans ATCC 27774, grown with nitrate as the terminal electron acceptor, which is the first example of a bacterioferritin from a strict anaerobic organism. This new bacterioferritin was isolated mainly as a 24-mer of 20 kDa identical subunits, containing 0.5 noncovalently bound heme and 2 iron atoms per monomer. Although its N-terminal sequence is significantly homologous with ferritins from other microorganisms and the ligands to the di-iron ferroxidase center are conserved, it is one of the most divergent bacterioferritins so far characterized. Also, in contrast to all other known bacterioferritins, its heme is not of the B type; its chromatographic behavior is identical to that of iron uroporphyrin. Thus, D. desulfuricans bacterioferritin appears to be the second example of a protein unexpectedly containing this heme cofactor, or a closely related porphyrin, after its finding in Desulfovibrio gigas rubredoxin:oxygen oxidoreductase ?Timkovich, R., Burkhalter, R. S., Xavier, A. V., Chen, L., and Le Gall, J. (1994) Bioorg. Chem. 22, 284-293. The oxidized form of the protein has a visible spectrum characteristic of low-spin ferric hemes, exhibiting a weak absorption band at 715 nm, indicative of bis-methionine heme axial coordination; upon reduction, the alpha-band appears at 550 nm and a splitting of the Soret band occurs, with two maxima at 410 and 425 nm. The heme center has a reduction potential of 140 +/- 10 mV (pH 7.6), a value unusually high compared to that of other bacterioferritins (ca. -200 mV).     

The fluidity of plasma membranes of Dictyostelium discoideum. The effects of polyunsaturated fatty acid incorporation assessed by fluorescence depolarization and electron paramagnetic resonance

The absorption spectrum of the hydrogenase from Chromatium, which contains four iron atoms and four atoms of acid-labile sulfide, in 80% dimethylsulfoxide or hexamethylphosphoramide suggests the presence of a single [4Fe-4S] cluster. The EPR spectra of the oxidized enzyme in air, argon or carbon monoxide are the same with signals centered at g = 2.01. The enzyme reduced by hydrogen is EPR silent. The EPR spectrum is consistent with a [4Fe-4S] cluster. Chromatium hydrogenase and the hydrogenase from Proteus vulgaris show relative stability towards denaturation by sodium dodecyl sulfate (SDS), urea, guanidine and organic solvents.     

The active centers of adenylylsulfate reductase from Desulfovibrio gigas. Characterization and spectroscopic studies

In order to utilize sulfate as the terminal electron acceptor, sulfate-reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO4) reductase plays one of the major roles, reducing AdoPSO4 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovibrio gigas. The protein is composed of two non-identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (approximately 400 kDa). It is an iron-sulfur, flavin-containing protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa. Low-temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g = 2.02 and only detectable below 20 K. This signal represented a minor species (0.10-0.25 spins/mol) and showed line broadening in the enzyme isolated from 57Fe-grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe-S center I) observable below 30 K and a concomitant decrease in intensity of the g = 2.02 signal were detected. Effects of chemical reductants (ascorbate, H2/hydrogenase-reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g = 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin-spin-coupled nature (observable below 20 K), indicating the presence of at least two iron-sulfur centers, (centers I and II). M?ssbauer studies on 57Fe-enriched D. gigas AdoPSO4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential less than or equal to 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin-type [4Fe-4S] cluster. Center I exhibits spectra with atypical M?ssbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin-type [4Fe-4S] cluster, suggesting a different structure and/or a distinct cluster-ligand environment.     

EPR signals from cytochrome c oxidase.

1. The major EPR signals from native and cytochrome c-reduced beef heart cytochrome c oxidase (EC 1.9.3.1) are characterized with respect to resonance parameters, number of components and total integrated intensity. A mistake in all earlier integrations and simulations of very anisotropic EPR signals is pointed out. 2. The so-called Cu2+ signal is found to contain at least three components, one "inactive" form and two nearly similar active forms. One of the latter forms, corresponding to about 20% of the total EPR detectable Cu, has not been observed earlier and can only be resolved in 35 GHz spectra. It is not reduced by cytochrome c and is thought to reflect some kind of inhomogeneity in the enzyme preparation. The 35 GHz spectrum of the cytochrome c reducible component shows a rhombic splitting and can be well simulated with g-values 2.18, 2.03 and 1.99. The origin of such a unique type of Cu2+ spectrum is discussed. 3. The low-spin heme signal in the oxidized enzyme (g = 3.03, 2.21, 1.45) is found to correspond closely to one heme and shows no signs of interaction with other paramagnetic centres. 4. The high-spin heme signals appearing in partly reduced oxidase are found to consist of at least three species, one axial and two rhombic types. An integration procedure is described that allows the determination of the total integral intensity of high-spin heme EPR signals only by considering the g = 6 part of the signals. In a titration with ascorbate and cytochrome c the maximum intensity of the g = 6 species corresponds to 23% of the enzyme concentration.     

Purification and characterization of a tungsten-containing formate dehydrogenase from Desulfovibrio gigas

An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein. Selenium was not detected. The UV/visible absorption spectrum of D. gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both M?ssbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with (57)Fe and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.     

M?ssbauer spectroscopic studies of the nature of centre X of photosystem I reaction centres from the cyanobacterium Chlorogloea fritschii

Reduced photosystem I samples, which give the electron paramagnetic resonance (EPR) signals associated with A, A and B, and A, B and X centres, have been studied using M?ssbauer spectroscopy. The M?ssbauer spectra obtained from each type of sample is different, which indicates that iron is associated with all three centres. The spectra are similar to those obtained from ferredoxins with 4Fe-4S centres and were fitted with oxidized and reduced components, the relative proportions depending on the degree of reduction of the sample as monitored by EPR. The sample which gave only the A EPR signal showed about 26% of the reduced component, the sample which gave A and B EPR signals showed about 48% of the reduced component, while the sample which gave A, B and X EPR signals showed about 65% of the reduced component. The measurements are consistent with X being a 4Fe-S4 centre.     

Evidence for a mu-oxo-bridged binuclear iron cluster in the hydroxylase component of methane monooxygenase. M?ssbauer and EPR studies

M?ssbauer and EPR studies of a highly active hydroxylase component of methane monooxygenase isolated from Methylosinus trichosporium OB3b are reported. The M?ssbauer spectra of the oxidized (as isolated) hydroxylase show iron in a diamagnetic cluster containing an even number of Fe3+ sites. The parameters are consistent with an antiferromagnetically coupled binuclear cluster similar to those of hemerythrin and purple acid phosphatases. Upon partial reduction of the hydroxylase, an S = 1/2 EPR spectrum with g values at 1.94, 1.86, and 1.75 (gav = 1.85) is observed. Such spectra are characteristic of oxo-bridged iron dimers in the mixed valent Fe(II).Fe(III) state. Further reduction leads to the appearance of a novel EPR resonance at g = 15. Comparison with an inorganic model compound for mu-oxo-bridged binuclear iron suggests that the g = 15 signal is characteristic of the doubly reduced state of the cluster in the protein. In this state, the M?ssbauer spectra exhibit two quadrupole doublets typical of high spin Fe2+, consistent with the Fe(II).Fe(II) form of the cluster. The spectral features of the iron center of the hydroxylase in three oxidation states are all similar to those reported for mu-oxo (or mu-hydroxo)-bridged binuclear iron clusters. Since no known monooxygenase contains such a cluster, a new oxygenase mechanism is suggested. Three different preparative methods yielded hydroxylases spanning a 9-fold range in specific activity, yet the same cluster concentration and spectral characteristics were observed. Thus, other parameters than those measured here have a major influence on the activity.     

Oxidation-reduction potentials of the hemes in cytochrome C3 from Desulfovibrio gigas in the presence and absence of ferredoxin by EPR spectroscopy.

1. Ferricytochrome c3 from D. gigas exhibits two low-spin ferric heme EPR resonances with gz-values at 2.959 and 2.853. Ferrocytochrome c3 is diamagnetic based on the absence of any EPR signals. 2. EPR potentiometric titrations result in the resolution of the two low-spin ferric heme resonances into two additional heme components representing in total the four hemes of the cytochrome, with EM values of -235 mV and -315 mV at heme resonance I and EM values of -235 mV and -306 mV at heme resonance II. 3. EPR spectroscopy has detected a significant diminution of intensity (approx. 60 p. 100) in the gx amplitude of ferricytochrome c3 in the presence of D. gigas ferredoxin II. The presence of ferredoxin II also causes a more negative shift in the EM of the second components of the signals at heme resonances I and II of cytochrome C3. Both observations suggest that an interaction has occurred between cytochrome C3 and ferredoxin II. 4. The results presented suggest that the heme ligand environment of ferricytochrome c3 from D. gigas is less perturbed and/or less asymmetric than environment for ferricytochrome c3 from D. vulgaris whose EPR behavior indicates the non-equivalence of all four hemes.     

Characterization of two soluble ferredoxins as distinct from bound iron-sulfur proteins in the photosynthetic bacterium Rhodospirillum rubrum.

In an earlier investigation (Shanmugam, K. T., Buchanan, B. B., and Arnon, D. I. (1972) Biochim. Biophys. Acta 256, 477-486) the extraction of ferredoxin from Rhodospirillum rubrum cells with the aid of a detergent (Triton X-100) and acetone revealed the existence of two types of ferredoxin (I and II) and led to the conclusion that both are membrane-bound. In the present investigation, ferredoxin and acid-labile sulfur analyses of photosynthetic membranes (chromatophores) and soluble protein extracts of the photosynthetic bacteria R. rubrum and Rhodopseudomonas spheroides showed that ferredoxins I and II are primarily components of the soluble protein fraction. After their removal, washed R. rubrum chromatophores were found to contain a considerable amount of tightly bound iron-sulfur protein(s), as evidenced by acid-labile sulfur and electron paramagnetic resonance analyses. Thus, like all other photosynthetic cells examined to date, R. rubrum cells contain both soluble ferredoxins and iron-sulfur proteins tightly bound to photosynthetic membranes. The molecular weights of ferredoxins I and II from photosynthetically grown R. rubrum cells were found to be 8,800 and 14,500, respectively. Using these molecular weights, the molar extinction coefficients at 390 nm for ferredoxins I and II were determined to be 30.3 and 17.2 mM-1 CM-1, respectively. Ferredoxin I contains 8 non-heme iron and 8 acid-labile sulfur atoms per molecule; ferredoxin II contains 4 non-heme iron and 4 acid-labile sulfur atoms per molecule. Ferredoxin I was found only in photosynthetically grown cells whereas ferredoxin II was present in both light- and dark-grown cells. Ferredoxin II from both light- and dark-grown cells has the same molecular weight (14,500) and absorption spectrum and has 4 iron and 4 acid-labile sulfur atoms per molecule. Low temperature electron paramagnetic resonance spectra of oxidized and photoreduced ferredoxins I and II from R. rubrum were recorded. The EPR spectrum of oxidized ferredoxin II exhibited a single resonance line at g = 2.012. Oxidized ferredoxin I, however, exhibited a spectrum that may arise from the superimposition of two resonance lines near g = 2.012. Photoreduced ferredoxin II displayed a rhombic EPR spectrum with a g value of 1.94. Photoreduced ferredoxin I exhibited a similar EPR spectrum at a temperature of 16 K, but when the temperature was lowered to 4.5 K the spectrum of ferredoxin I changed. This temperature-dependent spectrum may result from a weak spin-spin interaction between two iron-sulfur clusters. These results are consistent with the conclusion that R. rubrum ferredoxins I and II are, respectively, 8 iron/8 sulfur and 4 iron/4sulfur proteins.