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1.
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. 相似文献
2.
The combination of redox potentiometry with low temperature electron spin resonance (ESR) spectroscopy has led to further characterization of electron transfer components of Chromatium D. These include the readily buffer-soluble cytochromes c553 and c′ and the high-potential iron-sulfur protein in the isolated state and associated with the chromatophore membrane. Buffer-insoluble cytochrome c553, cytochro—me c555, bacteriochlorophyll and the primary electron acceptor have been characterized both in the chromatophore membrane and also in a sodium dodecylsulfate detergent-solubilized subchromatophore preparation. Two iron-sulfur proteins have been revealed which are present in the chromatophore membrane but are released on treatment with sodium dodecylsulfate. They have central g values at 1.90 and 1.94 and have estimated midpoint potentials at pH 7.4 (Em7·4) at +280 mV and ?100 mV, respectively, when associated with the chromatophore.In the membrane associated state the apparent Em of cytochrome c′ is approximately 200 mV more positive than the Em values reported for the free state; this implies either that the reduced form of cytochrome c′ binds to the membrane (or to a component therein) to a degree which is > 103 times greater than that of the oxidized form or that the Em shift results from membrane solvation. In the case of the high-potential iron-sulfur protein however, its Em when associated with the chromatophore membrane is similar to that reported in the isolated state. The light-induced oxidation of the high-potential iron-sulfur protein at room temperature appears to be linked only to the oxidation of cytochrome c555; it could serve as an electron pool in equilibrium with cytochrome c555 in the cyclic electron flow system.The redox component defined in the reduced state by its gy = 1.82 and gx = 1.62 ESR spectrum satisfies the following criteria for its identification as the primary electron acceptor of P883. (a) The Em7·4 value of the g = 1.82 component is ?120 ± 25mV. (b) At ?70 mV, where the g = 1.82 component is mainly oxidized in the dark, brief illumination at low temperature which causes the irreversible oxidation of one cytochrome c553 heme, also induces the permanent reduction of the g = 1.82 component; the extent of reduction after brief illumination, given by the g = 1.82 signal height, is the same as that induced chemically at ?270 mV showing it to be fully reduced by the receipt of a single electron. (c) At more positive potentials where cytochrome c553 is oxidized and is not involved in low-temperature reactions, the light-induced low-temperature kinetics of the g = 1.82 signal are reversible; the flash-induced g = 1.82 formation and subsequent dark decay are the same as those for the flash-induced P+883 (g = 2) formation and dark decay. We suggest that until a full physical-chemical characterization is completed this g = 1.82 component be designated “photoredoxin”. 相似文献
3.
Oriented multilayers made from beef heart and yeast mitochondria and submitochondrial particles were studied using electron paramagnetic resonance. EPR signals from membrane-bound iron-sulfur clusters and from a spin-coupled ubiquinone pair are highly orientation dependent, implying that these redox centers are fixed in the membrane at definite angles relative to the membrane plane. Typically the iron-iron axis (gz) of the binuclear iron-sulfur clusters is in the membrane plane. This finding is discussed in terms of the protein structure. the tetranuclear iron-sulfur clusters can have their gz axis either perpendicular or parallel to the membrane plane, but intermediate orientation was not observed. 相似文献
4.
Toshio Iwasaki Reinhard Kappl Gerhard Bracic Nobutaka Shimizu Daijiro Ohmori Takashi Kumasaka 《Journal of biological inorganic chemistry》2011,16(6):923-935
The crystal structure of the ISC-like [2Fe–2S] ferredoxin (FdxB), probably involved in the de novo iron-sulfur cluster biosynthesis (ISC) system of Pseudomonas putida JCM 20004, was determined at 1.90-Å resolution and displayed a novel tail-to-tail dimeric form. P. putida FdxB lacks the consensus free cysteine usually present near the cluster of ISC-like ferredoxins, indicating its primarily electron transfer role in the iron-sulfur cluster. Orientation-selective electron–nuclear double resonance spectroscopic analysis of reduced FdxB in conjunction with the crystal structure has identified the innermost Fe2 site with a high positive spin population as the nonreducible iron retaining the Fe3+ valence and the outermost Fe1 site as the reduced iron with a low negative spin density. The average g max direction is skewed, forming an angle of about 27.3° (±4°) with the normal of the [2Fe–2S] plane, whereas the g int and g min directions are distributed in the cluster plane, presumably tilted by the same angle with respect to this plane. These results are related to those for other [2Fe–2S] proteins in different electron transport chains (e.g. adrenodoxin) and suggest a significant distortion of the electronic structure of the reduced [2Fe–2S] cluster under the influence of the protein environment around each iron site in general. 相似文献
5.
A study of signals, light-induced at 77 K in O2-evolving Photosystem II (PS II) membranes showed that the EPR signal that has been attributed to the semiquinone-iron form of the primary quinone acceptor, Q?AFe, at g = 1.82 was usually accompanied by a broad signal at g = 1.90. In some preparations, the usual g = 1.82 signal was almost completely absent, while the intensity of the g = 1.90 signal was significantly increased. The g = 1.90 signal is attributed to a second EPR form of the primary semiquinone-iron acceptor of PS II on the basis of the following evidence. (1) The signal is chemically and photochemically induced under the same conditions as the usual g = 1.82 signal. (2) The extent of the signal induced by the addition of chemical reducing agents is the same as that photochemically induced by illumination at 77 K. (3) When the g = 1.82 signal is absent and instead the g = 1.90 signal is present, illumination at 200 K of a sample containing a reducing agent results in formation of the characteristic split pheophytin? signal, which is thought to arise from an interaction between the photoreduced pheophytin acceptor and the semiquinone-iron complex. (4) Both the g = 1.82 and g = 1.90 signals disappear when illumination is given at room temperature in the presence of a reducing agent. This is thought to be due to a reduction of the semiquinone to the nonparamagnetic quinol form. (5) Both the g = 1.90 and g = 1.82 signals are affected by herbicides which block electron transfer between the primary and secondary quinone acceptors. It was found that increasing the pH results in an increase of the g = 1.90 form, while lowering the pH favours the g = 1.82 form. The change from the g = 1.82 form to the g = 1.90 form is accompanied by a splitting change in the split pheophytin? signal from approx. 42 to approx. 50 G. Results using chloroplasts suggest that the g = 1.90 signal could represent the form present in vivo. 相似文献
6.
A single alkaline wash removes most of the succinic dehydrogenase activity from chromatophores of Rhodopseudomonas sphaeroides. Three iron-sulfur centers are also removed by this washing. Two of these are ferredoxin-like centers with electron paramagnetic resonance signals at gv = 1.94 and midpoint potentials of +50 and ?250 mV at pH 7. The third is a high-potential iron-sulfur protein type signal centered at g 2.01 and a midpoint potential of +80 mV at pH 7. These centers have very similar properties to those of the well-characterized mammalian succinic dehydrogenase and account for the majority of iron-sulfur centers observed in chromatophores. Because it is so easily removed, it is concluded that succinic dehydrogenase is located on the outer surface of the chromatophore membrane, a conclusion supported by the fact that removal of the enzyme does not interfere with the kinetics of light-induced electron flow, nor does it allow cytochrome c2 to escape from inside the chromatophore vesicles. 相似文献
7.
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 Em7.5 = +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 Em7.5 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 Em = ?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. 相似文献
8.
On the interpretation of electron paramagnetic resonance spectra of binuclear iron-sulfur proteins 总被引:1,自引:0,他引:1
A method is described for the interpretation of electron paramagnetic resonance spectra of reduced binuclear iron-sulfur proteins. The gy values for any protein can be analyzed so that both the symmetry and the extent of covalency at the paramagnetic site can be parameterized. These parameters can be related to the chemical composition of the paramagnetic center, the protein-dependent charge delocalization of the unpaired electron, and the geometric arrangement at the reduced iron atom. These analyses may ultimately be used to rationalize certain aspects of the redox potentials of the various iron-sulfur proteins. 相似文献
9.
The Rieske iron-sulfur center in the photosynthetic bacterium Rhodopseudomonas sphaeroides appears to be the direct electron donor to ferricytochrome c2, reducing the cytochrome on a submillisecond timescale which is slower than the rapid phase of cytochrome oxidation (). The reduction of the ferricytochrome by the Rieske center is inhibited by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) but not by antimycin. The slower (1–2 ms) antimycin-sensitive phase of ferricytochrome c2 reduction, attributed to a specific ubiquinone-10 molecule (Qz), and the associated carotenoid spectral response to membrane potential formation are also inhibited by UHDBT. Since the light-induced oxidation of the Rieske center is only observed in the presence of antimycin, it seems likely that the reduced form of Qz (QzH2) reduces the Rieske center in an antimycin-sensitive reaction. From the extent of the UHDBT-sensitive ferricytochrome c2 reduction we estimate that there are 0.7 Rieske iron-sulfur centers per reaction center.UHDBT shifts the EPR derivative absorption spectrum of the Rieske center from gy 1.90 to gy 1.89, and shifts the Em,7 from 280 to 350 mV. While this latter shift may account for the subsequent failure of the iron-sulfur center to reduce ferricytochrome c2, it is not clear how this can explain the other effects of the inhibitor, such as the prevention of cytochrome b reduction and the elimination of the uptake of H+II; these may reflect additional sites of action of the inhibitor. 相似文献
10.
Patrícia Raleiras Petra Kellers Peter Lindblad Stenbj?rn Styring Ann Magnuson 《The Journal of biological chemistry》2013,288(25):18345-18352
In nitrogen-fixing cyanobacteria, hydrogen evolution is associated with hydrogenases and nitrogenase, making these enzymes interesting targets for genetic engineering aimed at increased hydrogen production. Nostoc punctiforme ATCC 29133 is a filamentous cyanobacterium that expresses the uptake hydrogenase HupSL in heterocysts under nitrogen-fixing conditions. Little is known about the structural and biophysical properties of HupSL. The small subunit, HupS, has been postulated to contain three iron-sulfur clusters, but the details regarding their nature have been unclear due to unusual cluster binding motifs in the amino acid sequence. We now report the cloning and heterologous expression of Nostoc punctiforme HupS as a fusion protein, f-HupS. We have characterized the anaerobically purified protein by UV-visible and EPR spectroscopies. Our results show that f-HupS contains three iron-sulfur clusters. UV-visible absorption of f-HupS has bands ∼340 and 420 nm, typical for iron-sulfur clusters. The EPR spectrum of the oxidized f-HupS shows a narrow g = 2.023 resonance, characteristic of a low-spin (S = ½) [3Fe-4S] cluster. The reduced f-HupS presents complex EPR spectra with overlapping resonances centered on g = 1.94, g = 1.91, and g = 1.88, typical of low-spin (S = ½) [4Fe-4S] clusters. Analysis of the spectroscopic data allowed us to distinguish between two species attributable to two distinct [4Fe-4S] clusters, in addition to the [3Fe-4S] cluster. This indicates that f-HupS binds [4Fe-4S] clusters despite the presence of unusual coordinating amino acids. Furthermore, our expression and purification of what seems to be an intact HupS protein allows future studies on the significance of ligand nature on redox properties of the iron-sulfur clusters of HupS. 相似文献
11.
Tomoko Ohnishi 《BBA》1975,387(3):475-490
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 Em 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 gz = 2.03, gy = 1.94 and gx = 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 Em 7.2 value (?20±20 mV).Resolution of Centers N-3 and N-4 was achieved potentiometrically in submitochondrial particles. The component with Em 7.2 = ? 240±20 mV is defined as Center N-3 (gz = 2.10, (gy = 1.93?), gx = 1.87); the ?405±20 mV component as Center N-4 (gz = 2.11, (gy = 1.93?), gx = 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 Em 7.2 = ?260±20 mV. The multiplicity of peaks suggests the presence of at least two different ironsulfur centers having similar Em 7.2 values (?260±20 mV); hence, tentatively assigned as N-5 and N-6.Consistent with the individual Em 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 Em 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. Em 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 Em 7.2 values around -250 mV; Center N-2 exhibits a high Em 7.2 (?20±20 mV), while Center N-1a shows a very low Em 7.2 (?380±20 mV). 相似文献
12.
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. 相似文献
13.
Cristina Costa Miguel Teixeira Jean LeGall José J. G. Moura I. Moura 《Journal of biological inorganic chemistry》1997,2(2):198-208
An air-stable formate dehydrogenase, an enzyme that catalyzes the oxidation of formate to CO2, was purified from a sulfate-reducing organism, Desulfovibrio desulfuricans ATCC 27774. The enzyme has a molecular mass of approximately 150?kDa (three different subunits: 88, 29 and 16?kDa) and contains three types of redox-active centers: four c-type hemes, nonheme iron arranged as two [4Fe-4S]2+/1+ centers and a molybdenum-pterin site. Selenium was also chemically detected. The enzyme specific activity is 78 units per mg of protein. Mo(V) EPR signals were observed in the native, reduced and formate-reacted states. EPR signals related to the presence of multiple low-spin hemes were also observed in the oxidized state. Upon reduction, an examination of the EPR data under appropriate conditions distinguishes two types of iron-sulfur centers, an [Fe-S] center I (g max=2.050, g med=1.947, g min=1.896) and an [Fe-S] center II (g max=2.071, g med=1.926, g min=1.865). Mössbauer spectroscopy confirmed the presence of four hemes in the low-spin state. The presence of two [4Fe-4S]2+/1+ centers was confirmed, one of these displaying very small hyperfine coupling constants in the +1 oxidation state. The midpoint redox potentials of the enzyme metal centers were also estimated. 相似文献
14.
《BBA》1987,891(3):286-292
Photosystem I charge separation in a subchloroplast particle isolated from spinach was investigated by electron spin resonance (ESR) spectroscopy following graduated inactivation of the bound iron-sulfur centers by urea-ferricyanide treatment. Previous work demonstrated a differential decrease in iron-sulfur centers A, B and X which indicated that center X serves as a branch point for parallel electron flow through centers A and B (Golbeck, J.H. and Warden, J.T. (1982) Biochim. Biophys. Acta 681, 77–84). We now show that during inactivation the disappearance of iron-sulfur centers A, B, and X correlates with the appearance of a spin-polarized triplet ESR signal with |D| = 279·10−4 cm−1 and |E| = 39·10−4 cm−1. The triplet resonances titrate with a midpoint potential of +380 ± 10 mV. Illumination of the inactivated particles results in the generation of an asymmetric ESR signal with g = 2.0031 and ΔHpp = 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 ΔHpp = 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. 相似文献
15.
Jan I. Pedersen Helge Oftebro Tore Vänngård 《Biochemical and biophysical research communications》1977,76(3):666-673
An iron-sulfur protein has been isolated from bovine liver mitochondria and purified 140-fold on DEAE-cellulose and Sephadex G-100. During the isolation the protein was detected by its NADPH-cytochrome reductase activity in the presence of adrenal NADPH-ferredoxin reductase. The molecular weight of the protein (12,400), the optical spectrum (peaks at 414 nm and 455 nm which disappear upon reduction), and the EPR spectrum (gx = gy = 1.935 and gz = 2.02) were typical for a ferredoxin. In the presence of soluble adrenal cytochrome P450, ferredoxin reductase and NADPH, this protein could support the formation of pregnenolone from cholesterol. Under similar conditions, but in the presence of a cytochrome P450 solubilized from rat liver mitochondria, cholesterol was transformed into a more polar compound tentatively identified as 26-hydroxycholesterol. 相似文献
16.
The high-potential iron-sulfur protein (HiPIP) center of succinate dehydrogenase has an electron paramagnetic resonance (epr) signal in the oxidized form, centered at g = 2.01, and under certain conditions this epr signal is accompanied by absorbances at g = 2.04, g = 1.99, and g = 1.96. These absorbances have been attributed to a spin-spin interaction of paramagnetic species, the semiquinone form of ubiquinone being involved (Ruzicka et al., Proc. Nat. Acad. Sci. USA72, 2886). In the present work this magnetic interaction is studied further; it is concluded that of the three possible species (HiPIP, Flavin H and UQ?H (ubiquinone)) which may interact with UQ?H; a second UQ? most likely partner for the interaction. Nonetheless, the HiPIP center of succinate dehydrogenase also plays a role in the interaction by acting as a “magnetic relaxer” of one or both of the interacting UQ?Hs. The physiological reaction of that part of the ubiquinone pool associated with the succinate dehydrogenase (on the matrix side of the inner mitochondrial membrane) is UQH2 ? UQ?H + H+ + e?. This is in line with recent postulates of the mechanism of ubiquinone mediation in electron transfer. 相似文献
17.
Purified spinach nitrite reductase, a protein that contains siroheme, is characterized by absorption maxima in the visible region at 385 and 573 nm. On addition of the substrate nitrite, the bands shift to 360 and 570 nm. Dithionite also causes shifts in the maxima of the visible absorption region. Electron paramagnetic resonance studies show that the untreated enzyme contains a high-spin Fe3+ heme and that the addition of cyanide, an inhibitor that is competitive with nitrite, results in a spin-state change of the heme. Electron paramagnetic resonance analysis of the enzyme in the presence of dithionite or dithionite plus cyanide indicates the presence of a reduced iron-sulfur center with rhombic symmetry (g-values of 2.03, 1.94, and 1.91). In contrast, when the enzyme is treated with dithionite plus nitrite, the EPR spectrum of an NO-heme complex (g-values of 2.07 and 2.00) is observed. The presence of an iron-sulfur center has also been confirmed by chemical analyses of the nonheme iron and acid-labile sulfide in nitrite reductase. These results are discussed in terms of a mechanism for nitrite reduction that involves electron transfer between the iron-sulfur center and siroheme. 相似文献
18.
Roger C. Prince 《BBA》1983,723(2):133-138
Neutral and negatively charged dysprosium complexes are able to enhance the spin relaxation rate of the Rieske iron-sulfur cluster only when added from the cytochrome c2 side of the photosynthetic membrane, indicating that the Rieske cluster is asymmetrically placed in the membrane, nearer the cytochrome c2 side. The gz-axis of the Rieske cluster, taken to be the iron-iron axis of this binuclear cluster, lies in the membrane plane, as does the gy-axis. Appropriately, the gx-axis is orthogonal to the membrane plane. A comparison with a mammalian mitochondrial standard indicates that there are 0.65 ± 0.1 Rieske cluster per reaction center. This is in excellent agreement with previously determined estimates of the number of antimycin-binding sites, and binding sites for what is known phenomenologically as QZ, suggesting that there is one of each per ubiquinol-cytochrome c2 oxidoreductase. 相似文献
19.
Orientations of the active site chromophores of the mitochondrial cytochrome b-c1 complex incorporated into liposomes have been investigated in hydrated oriented multilayers of proteoliposome membranes using optical and epr spectroscopy. The hemes of cytochromes c1 and b were found to be oriented with the normal to their heme planes lying approximately in the plane of the proteoliposome membrane. Rieske's iron-sulfur center was oriented with the z-axis of the g tensor parallel to the plane of the membranes. It is concluded that the cytochrome b-c1 complex has a structural asymmetry which causes it to orient with respect to the lipid bilayer. 相似文献
20.
Rebecca Keller Jeanine de Keyzer Arnold J.M. Driessen Tracy Palmer 《The Journal of cell biology》2012,199(2):303-315
Membrane protein assembly is a fundamental process in all cells. The membrane-bound Rieske iron-sulfur protein is an essential component of the cytochrome bc1 and cytochrome b6f complexes, and it is exported across the energy-coupling membranes of bacteria and plants in a folded conformation by the twin arginine protein transport pathway (Tat) transport pathway. Although the Rieske protein in most organisms is a monotopic membrane protein, in actinobacteria, it is a polytopic protein with three transmembrane domains. In this work, we show that the Rieske protein of Streptomyces coelicolor requires both the Sec and the Tat pathways for its assembly. Genetic and biochemical approaches revealed that the initial two transmembrane domains were integrated into the membrane in a Sec-dependent manner, whereas integration of the third transmembrane domain, and thus the correct orientation of the iron-sulfur domain, required the activity of the Tat translocase. This work reveals an unprecedented co-operation between the mechanistically distinct Sec and Tat systems in the assembly of a single integral membrane protein. 相似文献