Cytochrome c interaction with the mitochondrial membrane: A spin label study

Horse-heart ferrocytochrome c has been labeled with N-(2,2,5,5-tetramethyl-3-pyrrolidinyl-1-oxyl) iodoacetamide at methionine-65. The paramagnetic resonance spectrum of labeled ferricytochrome c indicates a weak immobilization of the radical (τ_c = 9.3·10⁻¹⁰ sec) which becomes stronger upon binding of labeled cytochrome c to cytochrome c-depleted mitochondrial membranes (τ_c = 3.3·10⁻⁹ sec). The hyperfine coupling constant remains, however, unchanged (16.7 ± 0.1 gauss) indicating that the cytochrome c binding site is highly polar. The region where cytochrome c is bound to the membrane is insensitive to large variations of medium viscosity.     

Functional characterization of the mitochondrial cytochrome b-c1 complex: steady-state kinetics of the monomeric and dimeric forms

The QH2:cytochrome c oxidoreductase activity of the isolated bovine heart cytochrome b-c1 complex resolved into monomeric and dimeric form was titrated with three different inhibitors of electron transfer, antimycin, myxothiazol, and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). In all cases one inhibitor molecule per cytochrome c1 was found necessary to block completely the activity of both molecular forms of the enzyme. The antimycin-sensitive cytochrome c reduction catalyzed by the b-c1 complex was also studied as a function of increasing concentrations of either cytochrome c or quinol. Double-reciprocal plots of the activity of the monomeric enzyme were found linear either when the concentration of cytochrome c or of quinol derivatives, 2,3-dimetoxy-5-methyl-6-decyl-1,4-benzoquinol (DBH), and 2-methyl-3-undecyl-1,4-naphthoquinol (UNH), was changed. Cytochrome c reductase activity of the dimeric b-c1 complex also showed a linear Lineweaver-Burk plot as a function of cytochrome c concentrations. In contrast to the monomeric enzyme, however, dimers of the b-c1 complex express a clear nonlinear kinetic behavior toward quinol derivatives, with two apparent Km values differing approximately by one order of magnitude (about 3-4 and about 20-30 microM). At saturating quinol concentrations the activity of the dimeric enzyme becomes two to three times higher than that of monomers. The nonlinear kinetic plots were found to be the same at different temperatures and different cytochrome c concentrations. The data suggest that although the monomer of the b-c1 complex appears to be the functional unit of the enzyme, the dimer is more active. A regulatory role of the dimerization process resulting in an increase of the electrons flux through the enzyme is postulated.     

Molecular conversion between monomeric and dimeric states of the mitochondrial cytochrome b-c1 complex: isolation of active monomers

Bovine heart cytochrome b-c1 complex dispersed in 0.1% dodecylmaltoside, 10 mM Tris-HCl (pH 7.4), was subjected to filtration on Ultrogel AcA 34 columns. Apparent Mr values of about 400,000 and 170,000 were estimated for the enzyme-detergent complex in the presence and absence of 50 mM KCl, respectively. Similar Mr values (about 390,000 and 160,000) were obtained after sucrose gradient centrifugation of the b-c1 complex species isolated using Ultrogel filtration. Both species contained eight polypeptides, as in the original cytochrome b-c1 complex. The experiments suggest that the two species represent a dimer and a monomer of the b-c1 complex. The molecular conversion between the monomeric and dimeric state of the enzyme was found to be reversible. Both monomers and dimers of the b-c1 complex were competent to catalyze QH2:cytochrome c reductase activity with approximately the same maximal velocity. The finding that both molecular forms of the enzyme appear equally active does not support functional models based exclusively on a dimeric b-c1 complex.     

New insights on the cytochrome c oxidase proton pump.

Cytochrome c oxidase vesicles were used to show that, under appropriate experimental conditions: (1) no net deprotonation of the vesicular membrane or of the incorporated enzyme occurs during the oxidation of ferrocytochrome c; (2) the pH equilibration kinetics of a respiration-induced pH gradient across the bilayer are a simple function of the ohmic proton-conductance properties of the membrane; (3) a fairly constant stoichiometry (0.8-0.7) of the numbers of protons pumped per molecule of ferrocytochrome c oxidized, i.e. the H+/e- ratio, over a wide range of dioxygen molecules reduced (1-12) is observed.     

Subunit III of cytochrome c oxidase is expressed in Paracoccus denitrificans

Polyclonal antibodies have been obtained against a synthetic dodecapeptide identical to the aminoacid sequence 120-131 DSPIKDGVWPPE (inferred from its DNA sequence) of Paracoccus denitrificans cytochrome c oxidase subunit III. The antibodies had a titer higher than 1:10000 when tested against the antigen. These antibodies have been used to produce immunological evidence that, despite the fact that subunit III is not isolated with cytochrome c oxidase, it exists in Paracoccus denitrificans lysates. The antibodies did not show reactivity with bovine heart cytochrome c oxidase either by ELISA or immunoblotting. It was also shown that the antibodies react with a single polypeptide present in Paracoccus denitrificans cell lysates, having an apparent molecular weight close to that of subunit III of bovine heart oxidase.     

Isolation of tocopherol-binding proteins from the cytosol of smooth muscle A7r5 cells.

Cultured smooth muscle A7r5 cells were able to take up alpha-tocopherol (32 +/- 1.2 nmol/mg protein) the largest part of which (60%) was present in the cytosolic fraction. Using a tocopherol-based affinity chromatography and alpha-, beta-, gamma-, and delta-tocopherols as eluants, three polypeptides of molecular masses 81, 58 and 31 kDa were eluted. This preparation had alpha-[3H]tocopherol binding capability. The 58-kDa polypeptide could also be eluted by chromanol and the 81-kDa polypeptide could be eluted also by phytol. The 81-kDa polypeptide had the unique P-E-E-D-Q-X-Q-Y N-terminal sequence.     

A new method to determine DNA sugar conformation from the joint use of 2D and 3D NMR data

The use of sugar restraints has been proven essential for assessing DNAstructures through molecular modeling studies. We present a new methodcombining 2D (COSY and NOESY) and 3D (NOESY-NOESY) experiments, whereconstraints on either the phase angles or the difference between phase anglesof two residues are obtained from comparison of 2D NOE H1-H4intensities and 3D NOE intensities containing the H1-H4transfer. All experiments lead to restraints that match, proving the validityof the method.     

Two effects of electrical fields on chloroplasts

An electrical field across a suspension of Chenopodium chloroplasts stimulates the emission of delayed light during the time the field is on. This stimulation can be used to calculate the distance over which the electron moves in the untrapping process that gives the delayed light. An electrical field applied at the time of illumination gives a polarization to the suspension of chloroplasts that lasts for some seconds. This polarization is a new way to study delayed light and fluorescence from chloroplasts.