ABSTRACT
In a permeablized cell system, oxidized cyt c is able to induce caspase cascade whereas reduced cyt c cannot. In in vitro experiments, oxidized cyt c can promote H(2)O(2) generation. It is suggested that the redox state of cyt c might regulate the initiation of apoptosis via regulation of cellular ROS level.
Subject(s)
Caspases/metabolism , Cytochromes c , Models, Biological , Reactive Oxygen Species/metabolism , Signal Transduction/physiology , Apoptosis/physiology , Cell Line , Cytochromes c/chemistry , Cytochromes c/metabolism , Humans , Hydrogen Peroxide/metabolism , Mitochondria/metabolism , Oxidants/metabolism , Oxidation-Reduction , Oxygen/metabolismABSTRACT
The mitochondrial respiratory complex II, or succinate:ubiquinone oxidoreductase, is an integral membrane protein complex in both the tricarboxylic acid cycle (Krebs cycle) and aerobic respiration. The gene sequences of each complex II subunit were measured by RT-PCR. N-terminal sequencing work was performed to identify the mitochondrial targeting signal peptide of each subunit. Complex II was extracted from porcine heart and purified by the ammonium sulfate precipitation method. The sample was solubilized by 0.5% (w/v) sugar detergent n-decyl-beta-D-maltoside, stabilized by 200 mM sucrose, and crystallized with 5% (w/v) poly(ethylene glycol) 4000. Important factors for the extraction, purification and crystallization of mitochondrial respiratory complex II are discussed.
Subject(s)
Electron Transport Complex II/metabolism , Myocardium/metabolism , Amino Acid Sequence , Animals , Base Sequence , Crystallization , DNA Primers , Electron Transport Complex II/chemistry , Electron Transport Complex II/genetics , Electron Transport Complex II/isolation & purification , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Polymerase Chain Reaction , Swine , Transcription, GeneticABSTRACT
The mitochondrial respiratory Complex II or succinate:ubiquinone oxidoreductase (SQR) is an integral membrane protein complex in both the tricarboxylic acid cycle and aerobic respiration. Here we report the first crystal structure of Complex II from porcine heart at 2.4 A resolution and its complex structure with inhibitors 3-nitropropionate and 2-thenoyltrifluoroacetone (TTFA) at 3.5 A resolution. Complex II is comprised of two hydrophilic proteins, flavoprotein (Fp) and iron-sulfur protein (Ip), and two transmembrane proteins (CybL and CybS), as well as prosthetic groups required for electron transfer from succinate to ubiquinone. The structure correlates the protein environments around prosthetic groups with their unique midpoint redox potentials. Two ubiquinone binding sites are discussed and elucidated by TTFA binding. The Complex II structure provides a bona fide model for study of the mitochondrial respiratory system and human mitochondrial diseases related to mutations in this complex.
Subject(s)
Cell Respiration/physiology , Electron Transport Complex II/chemistry , Electron Transport/physiology , Intracellular Membranes/metabolism , Mitochondria/metabolism , Animals , Binding Sites/physiology , Crystallography, X-Ray , Electron Transport Complex II/metabolism , Enzyme Inhibitors/pharmacology , Flavoproteins/chemistry , Iron-Sulfur Proteins/chemistry , Membrane Proteins/chemistry , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Protein Structure, Tertiary/physiology , Protein Subunits/chemistry , Sequence Homology, Amino Acid , Succinic Acid/metabolism , Sus scrofa , Ubiquinone/metabolismABSTRACT
Bacteriorhodopsin (BR) films oriented by an electrophoretic method are deposited on a transparent conductive ITO glass. A counterelectrode of copper and gelose gel is used to compose a sandwich-type photodetector with the structure of ITO/BR film/gelose gel/Cu. A single 30-ps laser pulse and a mode-locked pulse train are respectively used to excite the BR photodetector. The ultrafast falling edge and the bipolar response signal are measured by the digital oscilloscope under seven different time ranges. Marquardt nonlinear least squares fitting is used to fit all the experimental data and a good fitting equation is found to describe the kinetic process of the photoelectric signal. Data fitting resolves six exponential components that can be assigned to a seven-step BR photocycle model: BR-->K-->KL-->L-->M-->N-->O-->BR. Comparing tests of the BR photodetector with a 100-ps Si PIN photodiode demonstrates that this type of BR photodetector has at least 100-ps response time and can also serve as a fast photoelectric switch.
