Identification of amino acid residues essential for reconstitutive activity of subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides

Tso, Shih-Chia; Yin, Ying; Yu, Linda

doi:10.1016/j.bbabio.2006.06.010

“…Indeed, the recent structural studies of a supercomplex formed from complexes I and III indicate that the matrix part of complex III is required for its association with complex I (14). The matrix region of Complex III may also be important for stabilization of the complex itself as bc 1 complexes without core proteins are generally less stable and have lower activity (15). If the interaction between the matrix enzymes and the bc 1 complex exists, this region would be a good choice for the contact.…”

mentioning

confidence: 99%

Crosstalk between Mitochondrial Malate Dehydrogenase and Cytochrome bc1 Complex

Wang

¹

,

Yu

²

2009

Biophysical Journal

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The interactions between the mitochondrial cytochrome bc 1 complex and matrix-soluble proteins were studied by a precipitation pulldown technique. Purified, detergent-dispersed bc 1 complex was incubated with mitochondrial matrix proteins followed by dialysis in the absence of detergent. The interacting protein(s) was co-precipitated with bc 1 complex upon centrifugation. One of the matrix proteins pulled down by bc 1 complex was identified as mitochondrial malate dehydrogenase (MDH) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and confirmed by Western blotting with anti-MDH antibody. Using a cross-linking technique, subunits I, II (core I and II), and V of the bc 1 complex were identified as the interacting sites for MDH. Incubating purified MDH with the detergent dispersed bc 1 complex results in an increase of the activities of both the bc 1 complex and MDH. The effect of the bc 1 complex on the activities of MDH is unidirectional (oxaloacetate 3 malate). These results suggest that the novel cross-talk between citric acid cycle enzymes and electron transfer chain complexes might play a regulatory role in mitochondrial bioenergetics.

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“…Indeed, the recent structural studies of a supercomplex formed from complexes I and III indicate that the matrix part of complex III is required for its association with complex I (14). The matrix region of Complex III may also be important for stabilization of the complex itself as bc 1 complexes without core proteins are generally less stable and have lower activity (15). If the interaction between the matrix enzymes and the bc 1 complex exists, this region would be a good choice for the contact.…”

mentioning

confidence: 99%

Cross-talk between Mitochondrial Malate Dehydrogenase and the Cytochrome bc1 Complex

Wang

¹

,

Yu

²

2010

Journal of Biological Chemistry

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The interactions between the mitochondrial cytochrome bc 1 complex and matrix-soluble proteins were studied by a precipitation pulldown technique. Purified, detergent-dispersed bc 1 complex was incubated with mitochondrial matrix proteins followed by dialysis in the absence of detergent. The interacting protein(s) was co-precipitated with bc 1 complex upon centrifugation. One of the matrix proteins pulled down by bc 1 complex was identified as mitochondrial malate dehydrogenase (MDH) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and confirmed by Western blotting with anti-MDH antibody. Using a cross-linking technique, subunits I, II (core I and II), and V of the bc 1 complex were identified as the interacting sites for MDH. Incubating purified MDH with the detergent dispersed bc 1 complex results in an increase of the activities of both the bc 1 complex and MDH. The effect of the bc 1 complex on the activities of MDH is unidirectional (oxaloacetate 3 malate). These results suggest that the novel cross-talk between citric acid cycle enzymes and electron transfer chain complexes might play a regulatory role in mitochondrial bioenergetics.The mitochondrial cytochrome bc 1 complex catalyzes electron transfer from ubiquinol to cytochrome c with the concomitant translocation of protons across the membrane to generate a proton gradient and membrane potential for ATP synthesis (1). Three-dimensional structures of mitochondrial bc 1 complexes from various sources have become available in recent years (2-11). All mitochondrial cytochrome bc 1 complexes contain three essential subunits that house four redox prosthetic groups and seven to eight non-redox prosthetic groups containing supernumerary subunits. The three essential subunits are: the cytochrome b subunit, housing two b-type hemes (b H and b L ); the cytochrome c 1 subunit, housing one c-type heme (c 1 ); the Rieske iron-sulfur protein (ISP), 2 housing a high potential [2Fe-2S] cluster.Although the structural and functional studies of the mitochondrial bc 1 complex have been intensive, few studies of the interactions between the bc 1 complex and the soluble matrix enzymes have been available. Complex II (succinate-ubiquinone oxidoreductase) is the only enzyme that participates in both the citric acid cycle and the electron transport chain (12). Complex II is composed of a succinate dehydrogenase, which is a citric acid cycle enzyme, and a membrane-anchoring protein fraction, which houses ubiquinone. Complex II catalyzes the oxidation of succinate to fumarate and the reduction of ubiquinone to ubiquinol. Other enzymes of citric acid cycle are known to couple to the electron transfer chain through their product NADH at complex I (13). Structurally the mitochondrial bc 1 complex can be divided into three regions: matrix, transmembrane, and the intermembrane space regions. The matrix region contains subunits I (core I), II (core II), VI, IX, and the N-terminal part of subunits V (ISP) and VII (2, 4). Although this region contains the m...

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“…When purified recombinant subunit IV was added to the three-subunit core complex, the bc 1 activity was restored to the level of the wild-type complex, indicating that recombinant subunit IV can properly assemble into the cytochrome bc 1 complex [8]. The availability of reconstitutively active, three-subunit core complex and recombinant subunit IV enables us to use an in vitro reconstitution approach to identify the amino acid residues of subunit IV involved in interaction with the core complex [9, 10]. Residues 81–84, with a sequence of YRYR [10], and residues 86–109, which comprise the only transmembrane helix in subunit IV [9], are essential.…”

Section: Introductionmentioning

confidence: 99%

“…The availability of reconstitutively active, three-subunit core complex and recombinant subunit IV enables us to use an in vitro reconstitution approach to identify the amino acid residues of subunit IV involved in interaction with the core complex [9, 10]. Residues 81–84, with a sequence of YRYR [10], and residues 86–109, which comprise the only transmembrane helix in subunit IV [9], are essential. It has been suggested that subunit IV is incorporated into the bc 1 complex through its transmembrane helix and then interacts with the core subunits through residues 81–84 to restore the bc 1 activity to the level of the wild-type complex [10].…”

Section: Introductionmentioning

confidence: 99%

“…Residues 81–84, with a sequence of YRYR [10], and residues 86–109, which comprise the only transmembrane helix in subunit IV [9], are essential. It has been suggested that subunit IV is incorporated into the bc 1 complex through its transmembrane helix and then interacts with the core subunits through residues 81–84 to restore the bc 1 activity to the level of the wild-type complex [10]. However, it is not known how this interaction can restore the bc 1 activity.…”

Section: Introductionmentioning

confidence: 99%

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Generation, characterization and crystallization of a cytochrome c1-subunit IV fused cytochrome bc1 complex from Rhodobacter sphaeroides

Su

¹

,

Esser

²

,

Xia

³

et al. 2012

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Cytochrome bc1 complex catalyzes the reaction of electron transfer from ubiquinol to cytochrome c (or cytochrome c2) and couples this reaction to proton translocation across the membrane. Crystallization of the R. sphaeroides bc1 complex resulted in crystals containing only three core subunits. To mitigate the problem of subunit IV being dissociated from the three-subunit core complex during crystallization, we recently engineered an R. sphaeroides mutant in which the N-terminus of subunit IV was fused to the C-terminus of cytochrome c1 with a 14-glycine linker between the two fusing subunits, and a 6-histidine tag at the C-terminus of subunit IV (c1-14Gly-IV-6His). The purified fusion mutant complex shows higher electron transfer activity, more structural stability, and less superoxide generation as compared to the wild-type enzyme. Preliminary crystallization attempts with this mutant complex yielded crystals containing four subunits and diffracting x-rays to 5.5 Å resolution.

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Identification of amino acid residues essential for reconstitutive activity of subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides

Cited by 8 publications

References 16 publications

Crosstalk between Mitochondrial Malate Dehydrogenase and Cytochrome bc1 Complex

Crosstalk between Mitochondrial Malate Dehydrogenase and Cytochrome bc1 Complex

Cross-talk between Mitochondrial Malate Dehydrogenase and the Cytochrome bc1 Complex

Generation, characterization and crystallization of a cytochrome c1-subunit IV fused cytochrome bc1 complex from Rhodobacter sphaeroides

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