The Rieske FeS protein

After suggestions on an iron-sulphur protein in the 6c, complex [215], this subunit was isolated and characterised by Rieske et al. [216,218], but in a form that was not active in reconstitution. Isolation in a reconstitutively active form was pioneered by Racker et al. [219], who showed that a soluble oxidation factor was required for activity. Subsequently, Trumpower and Edwards [220] purified oxidation factor and identified it as a form of the FeS protein that is active in reconstitution. An excellent review on the structure and function of the FeS protein is available [221]. 

The iron-sulphur protein has an apparent molecular weight of 24-25 000. Li et al. [222] were able to split it into two parts by limited proteolysis. While the native protein is soluble only in the presence of detergent, a 16 kDa water-soluble fragment was released, which apparently contains the FeS centre. From this it appears that the Rieske protein is amphiphilic (cf., cytochrome c,), containing a membrane anchor and a catalytic centre in different domains. 

The iron-sulphur centre is probably of the 2Fe-2S type [191,223]. It is a one-electron donor/acceptor with of approx. 280 mV in mitochondria (pH independent below pH 8 [224,225]). It exhibits an EPR spectrum in the reduced state that is somewhat anomalous for 2Fe-2S clusters (see Ref. 221). This, as well as the high midpoint redox potential, suggest that the iron ligands may be less electronegative than the four cysteine sulphurs of the plant ferredoxin model (see Ref. 226). The EPR spectrum of the FeS cluster is affected by the redox state of ubiquinone 

The FeS protein functions as a ubiquinohferricytochrome Cj oxidoreductase. It also has a central role in the coupled oxidation of semiquinone by ferricytochrome b (see Refs. 179, 221, 229 and below). 

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This was the first complete structure of the bc complex. The structure provided information about all 11 subunits and revealed that subunit 9, the mitochondrial targeting presequence of ISP, exists between two core subunits, which are most likely a mitochondrial targeting presequence peptidase. We have solved the structures of the bc complex in two different crystal forms. Surprisingly, the conformation of the Rieske FeS protein was totally different between two crystal forms, and this provided a crucial insight of the electron bifurcation mechanism at the Qp site (see Section II,F). 

Figure 18 Schematic structure of the cytochrome bc complex from mitochondria. The struemre of the complex from purple photosynthetic bacteria is thought to be similar. The pathway of electron and proton transfer (modified Q-cycle) is overlaid on the schematic structure. Movement of the Rieske FeS protein is shown by the semitransparent yellow areas ...

The core proteins and the Rieske FeS protein can be dissociated from Triton X-lOO-solubilised cytochrome c reductase in concentrated NaCl. The cytochrome core was isolated from the dissociated Complex III of Neurospora by gel filtration [230]. It retains most of the hydrophobic character of the parent protein and seems to correspond to the membrane-embedded part of Complex III. 

Fig. 3.10. Topography of Complex III. Complex III is a dimer in the two-dimensional crystal form studied by electron microscopy. The shape of the membrane-bound enzyme particle was resolved by image reconstruction of micrographs [230]. The location of various components was predicted by comparing crystals of Complex III with those of a subcomplex lacking the Rieske FeS protein and the core proteins [222,231]. The schematic figure is adapted from Li et al. [222].

Efforts toward developing synthetic models for the Rieske Fe-S centers focussed initially on preparing Fe2S2 cores with non-thlolate ligands, and have centered on nitrogenous ligands since the realization of their probable occurrence in the Rieske protein. In addition to the [Fe2S2(0Ar) ] " ions (Ar = aryl)... 

Complex III crystallizes in two distinct conformations (not shown). In one, the Rieske Fe-S center is close to its electron acceptor, the heme of cytochrome c, but relatively distant from cytochrome b and the QH2-binding site at which the Rieske Fe-S center receives electrons. In the other, the Fe-S center has moved away from cytochrome c, and toward cytochrome b. The Rieske protein is thought to oscillate between these two conformations as it is reduced, then oxidized. 

Molik, S., Karnauchov, I., Weidlich, C., Herrmann, R. G., and Klosgen, R. B. (2001). The Rieske Fe/S protein of the cytochrome be,// complex in chloroplasts missing link in the evolution of protein transport pathways in chloroplasts J. Biol. Chem. 276, 42761-42766. 

As is indicated in Table 5-3, P680, P70o> the cytochromes, plastocyanin, and ferredoxin accept or donate only one electron per molecule. These electrons interact with NADP+ and the plastoquinones, both of which transfer two electrons at a time. The two electrons that reduce plastoquinone come sequentially from the same Photosystem II these two electrons can reduce the two >-hemes in the Cyt b(f complex, or a >-heme and the Rieske Fe-S protein, before sequentially going to the /-heme. The enzyme ferre-doxin-NADP+ oxidoreductase matches the one-electron chemistry of ferredoxin to the two-electron chemistry of NADP. Both the pyridine nucleotides and the plastoquinones are considerably more numerous than are other molecules involved with photosynthetic electron flow (Table 5-3), which has important implications for the electron transfer reactions. Moreover, NADP+ is soluble in aqueous solutions and so can diffuse to the ferredoxin-NADP+ oxidoreductase, where two electrons are transferred to it to yield NADPH (besides NADP+ and NADPH, ferredoxin and plastocyanin are also soluble in aqueous solutions). 

The Cyt bIf complex is the only electron transport complex for which the transmembrane organization of all its subunits is established. This membrane-spanning complex that functions as an intermediate electron transport complex between PS II and PS I, and translocates protons across the membrane from the stroma to the lumen, contains 4 proteins Cyt / (33 kDa), Cyt 6-563 (23 kDa), the Rieske Fe-S protein (20 kDa) and the unnamed 17 kDa protein. 

The Cyt b-f complex contains the redox components Cyt /, Cyt 6-563 and the Rieske Fe-S protein, which in spinach have been identified as polypeptides of 33, 23 and 20 kDa respectively [95,98]. The spinach complex contains in addition a polypeptide of 17 kDa with no known redox function. The reported sizes of these polypeptides estimated by SDS-gel electrophoresis vary somewhat between different laboratories, presumably beca.use of slightly different electrophoretic procedures. The estimated size of the Cyt / polypeptide varies between different plants, even when analysed in the same electrophoresis system, although the gene sequences predict polypeptides of very similar relative molecular mass. 

As with all the other photosynthetic membrane complexes, the genes for the components of the cytochrome complex are distributed between the nuclear and chlo-roplast genomes of higher plants. The chloroplast genes for the Cyt /, Cyt 6-563 and 17 kDa polypeptides have been extensively characterized, but the nuclear gene(s) for the Rieske Fe-S protein have not yet been isolated. 

The gene(s) for the Rieske Fe-S protein has not been isolated, but its location in nuclear DNA is predicted from the synthesis of a precursor form from poly(A) RNA isolated from spinach [109]. cDNA clones containing the coding sequence of the Rieske Fe-S protein should be forthcoming shortly. 

The Rieske Fe-S protein is synthesized on cytoplasmic ribosomes to give a 27 kDa precursor form [109]. This precursor is presumably required for targetting to the chloroplasts. There is no information on the regulation of expression of the gene for the Rieske Fe-S protein, nor on the assembly of the protein into the Cyt b-f complex. 

As indicated, above, the two Z -hemes of the Cyt b f complex provide a pair of reacting sites spanning the thylakoid membrane, one near the stromal side and the other near the lumenal side of the thylakoid membrane. The plastohydroquinone is first oxidized by the Rieske FeS to a semiquinone, which is then oxidized by cytochrome/, which then releases the electron to the copper protein plastocyanin. After loss of one electron by the plastohydroquinone, the resulting semiquinone loses an electron to the two fc-hemes in series. The Z -hemes operate in the so-called Q-cycle, similar to that in the mitochondrial or bacterial cytochrome bc complex, and provide a translocation of additional protons across the membrane into the lumenal space. Discussion of the cytochrome b(,f complex and the Q-cycle will be presented in Chapter 35. 

J Steppuhn, C Rother, J Hermans, J Salnikow, G Hauska and RG Herrmann (1987) The complete amino acid sequence of the Rieske Fe-S precursor protein from spinach chloroplasts deduced from cDNA analysis. Mol Gen Genet 210 171-177... 

RD Britt, K Sauer, MP Klein, DB Knaff, A Kriauciunas, C-A Yu, L Yu and R Malkin (1991) Electron spin echo modulation spectroscopy supports the suggested coordination of two histidine ligands to the Rieske Fe-S centers of the cytochrome b f complex of spinach and cytochrome bc complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R26, and bovine heart mitochondria. Biochemistry 30 1892-1901 R Gurbiel, T Ohnishi, DE Robertson, F Daldal and BM Hoffman (1991) Q-band ENDOR spectra of the Rieske protein from Rhodobacter capsulatus ubiquinol-cytochrome c oxidoreductase show two histidines coordinated to the [2Fe2S] cluster. Biochemistry 30 11579-11584... 

Hypothesis 2 Hydrophobic Association Within Complex III of the Hydrophobic (FeS) Tip of the Rieske Iron Protein with the Hydrophobic Ubiquinol-containing Qo Site Causes Extension and Damping of Internal Chain Dynamics in the Tether of the Iron Protein... 

Consilient Mechanisms for Electron Transfer/Proton Transport Reside in the Movement of the Rieske Iron Protein FeS Center... 

Is the Tether of the Rieske Iron Protein a Free Standing (Kinetically Free) Chain When the FeS Center Is at the... 

Figure 8.14. Stereo view of the monomer of Complex III the cytochrome bci complex of chicken. Shown are ligands plus the Rieske Iron Protein, where the inhibitors stigmatellin and antimycin are used to show FeS center relocation on reduction. The Rieske Iron Protein anchors in one monomer and then reaches across to accept an electron at its FeS center from the Qo site of the second monomer. (A) With inhibitors, the FeS center is in position, with the tip of the globular component of the Rieske Iron Protein pointing directly downward at the level of the Qo site, where it is to be reduced by the second component of the functional homodimer. (Prepared using the crystallographic results of Zhang et al. as...

Figure 8.15. Stereo view of yeast cytochromes b and Cl of Complex III showing Qo and heme Ci binding sites for the FeS center of the Rieske Iron Protein. Neutral residues are given in light gray, aromatic residues in black, other hydrophobic residues in gray, and charged residues in white in order to show visually the relative hydrophobicity of the two sites. Note at the Qo site that ubiquinol would reside at the base of a hydrophobic pit into which the hydrophobic FeS tip of the Rieske Iron Protein fits (see Figure 8.16), whereas the heme Cj site is not as hydrophobic. Also note that the hydrophobic residues L263 and V264...

S Is the Tether Contracted When the FeS Center of the Rieske Iron Protein Resides at the Heme Ci Site ... 

Application of the impure bef complex to the hydroxyapatite column yielded a similar profile as in Fig. 2b, however with a predominant cyt.bef-peak (not shown). The four subunits of about 38, 24, 19 and 15 kDa (Table I) are likely to be cytochrome f- and cytochrome be-binding subunit, the Rieske Fe-S-protein and subunit IV, respectively, consistent with the subunit composition of other cyanobacterial cyt.bef-complexes. The presence of b-type and c-type cytochromes in a ratio of 2 1 was further confirmed by reduced-minus-oxidised difference spectra (not shown). 

Mechanistically, the cyt bej complex is thought to contain two distinct catalytic domains located on each side of the membrane (8,9,10). The quinol oxidation site (called Qz in bacterial and Qo in mitochondrial systems) is on the outer side of the membrane. It converts a quinol molecule to a quinone by transferring an electron to the Rieske FeS center and another to the lower potential cyt b heme ( l) This second electron is subsequently transferred to the cyt bn which then reduces a quinone trapped at the quinone reduction site (called Qc in bacterial, Qi in mitochondrial systems) located in the vicinity of the inner negative face of the membrane (8,14). Several classes of inhibitors are known to affect the reactions catalyzed at these active sites of the cyt bci complex (11). Myxothiazol, mucidin and stigmatellin interfere with the electron transfer between ubiquinol, Rieske FeS protein and cyt bi at the Qz site (28). Although stigmatellin also affects the Photosystem II of... 

The cytochrome b-f complex of the chloroplast thylakoid membrane operates as a plastoquinol-plastocyanin oxidoreductase and is thus analogous to the mitochondrial cytochrome bc complex with which it bears a large amount of structural and functional similarity. It consists in all higher plants of four components, which are cytochrome f, cytochrome b-563 the Rieske Fe-S protein, and a 17kDa... 

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