Spatial Organization of the Redox Active Centers in the Bovine Heart Ubiquinol-cytochrome c Oxidoreductase

Site-directed mutations of conserved residues in the domain binding the 2Fe-2S cluster of the Rieske subunit of the ubiquinol:cytochrome c2 oxidoreductase (bc1 complex) of Rhodobacter sphaeroides have been constructed. The substitution of aspartate for glycine at position 133 in the Rb. sphaeroides sequence (mutant FG133D), which mimicked a mutation previously isolated and characterized in yeast by Gatti et al. [Gatti, D.L., Meinhardt, S.W., Ohnishi, T., & Tzagoloff, A. (1989) J. Mol. Biol. 205, 421-435], allowed more detailed studies of thermodynamic behavior and the kinetics of the ubiquinol:cytochrome c2 oxidoreductase on flash activation of the photosynthetic chain. The impaired catalysis in this mutant complex is localized to the quinol oxidizing site. The apparent second-order rate constant for reduction of cytochrome bH via the quinol oxidizing site is about 20-fold lower than that of the wild-type and correlates with its apparent activation barrier being increased relative to that of the wild-type. Substitutions for the cysteines and a histidine which are conserved in the putative 2Fe-2S binding domain of the Rieske subunit selectively knock out the 2Fe-2S cluster and quinol oxidizing activity, while leaving the cytochromes and other catalytic sites essentially intact. Reversion properties of these strains are consistent with the mutated residues being essential.(ABSTRACT TRUNCATED AT 250 WORDS)

mentioning

confidence: 54%

Site-directed mutations of conserved residues of the Rieske iron-sulfur subunit of the cytochrome bc1 complex of Rhodobacter sphaeroides blocking or impairing quinol oxidation

Doren

Gennis²,

Barquera³

et al. 1993

“…This effect is also seen in the whole cell preparations used in this work (data not shown). The effect of DyEDTA on the line width of an intrinsic center is effective over a greater distance than its effect on the spin relaxation of the intrinsic center (an H versus an r4 to r6 distance dependence; Blum et al, 1981;Ohnishi et al, 1989). For this reason, we were unable to assign the membrane sidedness of the [3Fe-4S] cluster of the DmsB-C 102S mutant from the line-broadening effect.…”

Section: Resultsmentioning

confidence: 94%

“…It would be interesting Gadolinium(III) and dysprosium(III) have been used as effective paramagnetic probes in a number of proteins containing electron paramagnetic resonance (EPR) visible species. These include the photoreaction center of PSII in Rhodobacter sphaeroides (Innes & Brudvig, 1989), the bovine cytochrome bc\ complex (Ohnishi et al, 1989), and Rhodobacter capsulatus cytochrome bc\ (Meinhardt & Ohnishi, 1992). These probes are most effective in systems where there are magnetically isolated paramagnetic species present in the protein being studied.…”

mentioning

confidence: 99%

Topological characterization of Escherichia coli DMSO reductase by electron paramagnetic resonance spectroscopy of an engineered iron-sulfur [3Fe-4S] cluster

Rothery

Weiner²

1993

We have applied the technique of distance estimations using the exogenous paramagnetic probe dysprosium (III) complexed with EDTA (DyEDTA) to study the topology of Escherichia coli dimethyl sulfoxide reductase (DmsABC) in situ in cytoplasmic membrane and whole cell preparations. The electron transfer subunit (DmsB) of this enzyme contains four [4Fe-4S] clusters and has complex EPR properties making it unsuitable for studies utilizing exogenous paramagnetic probes. We have utilized a mutant of DmsABC in which one of the [4Fe-4S] clusters of DmsB has been changed to a [3Fe-4S] cluster [Rothery, R. A., & Weiner, J. H. (1991) Biochemistry 30, 8296-8305]. This mutant (DmsB-C102S) has a single magnetically isolated EPR visible [3Fe-4S] cluster in its fully oxidized state, making it suitable for studies using DyEDTA as paramagnetic probe. We have studied the effect of DyEDTA on the microwave power saturation properties of the EPR signal of the DmsB-C102S mutant. DyEDTA enhances the spin relaxation of the [3Fe-4S] signal in everted membrane vesicles. It has a smaller effect on the spin relaxation of the [3Fe-4S] signal in whole cell preparations. We conclude that the [3Fe-4S] cluster of the DmsB-C102S mutant is located on the inside of the cytoplasmic membrane. We have estimated the distance of this center from the surface of the DmsAB dimer to be approximately 21 A, close to the cytoplasmic-side membrane surface level, by calibrating the DyEDTA effect using a myoglobin nitroxide standard.

“…The CD couplet originating from the exciton coupling of the two hemes overlaps and obscures the CD signals that each b heme possesses independently of the other. These latter CD features derive from the coupling of the b hemes with nearby aromatic residues of the apoprotein (Degli Esposti et al, 1987Esposti et al, , 1989a and are less intense in the reduced than in the oxidized cytochromes, presumably because the dipole strength of the reduced heme is smaller than that of the oxidized heme (Table 1; from EPR and linear dichroism spectroscopy (Erecinska & Wilson, 1979;Palmer, 1985;Von Jagow et al, 1986;Ohnishi et al, 1989) and from the analysis of the primary sequence deduced from the gene from diverse species (Saraste, 1984;Crofts et al, 1987Crofts et al, , 1992Degli Espos ti et al, 1989bTron et al, 1991). It is commonly agreed that the two b hemes are buried within the transmembrane portion of the protein and are coordinated to four invariant histidines, Hiss2, HÍS95, Hisis3, and HÍS197 (Figure 4).…”

Section: Resultsmentioning

confidence: 99%

Application of exciton coupling theory to the structure of mitochondrial cytochrome b

Palmer¹,

Esposti²

1994

The circular dichroism of the reduced bis-heme mitochondrial cytochrome b in the Soret region has been analyzed using exciton theory. The theory was applied to a geometric model that reflects the current consensus of the structure of this membrane protein [Degli Esposti, M., De Vries, S., Crimi, M., Ghelli, A., Patarnello, T., & Meyer, A. (1993) Biochim. Biophys. Acta 1143, 243-271]. The analysis suggests that the iron-iron distance is about 19.5 A, with a possible range of 17-23 A; the two hemes appear to be canted at 45 degrees with respect to one another. It appears that the two hemes undergo a relative angular displacement upon reduction, suggesting some flexibility in the protein structure. Some applications of this analysis to other bis-heme proteins are considered.