ABSTRACT
Recent evidence suggests that the activity of mitochondrial oxidative phosphorylation complexes (MOPCs) is modulated at multiple sites. Here, a method of optically monitoring electron distribution within and between MOPCs is described using a center-mounted sample in an integrating sphere (to minimize scattering effects) with a rapid-scanning spectrometer. The redox-sensitive MOPC absorbances (â¼465-630 nm) were modeled using linear least squares analysis with individual chromophore spectra. Classical mitochondrial activity transitions (e.g., ADP-induced increase in oxygen consumption) were used to characterize this approach. Most notable in these studies was the observation that intermediates of the catalytic cycle of cytochrome oxidase are dynamically modulated with metabolic state. The MOPC redox state, along with measurements of oxygen consumption and mitochondrial membrane potential, was used to evaluate the conductances of different sections of the electron transport chain. This analysis then was applied to mitochondria isolated from rabbit hearts subjected to ischemia/reperfusion (I/R). Surprisingly, I/R resulted in an inhibition of all measured MOPC conductances, suggesting a coordinated down-regulation of mitochondrial activity with this well-established cardiac perturbation.
Subject(s)
Mitochondria/chemistry , Optics and Photonics/methods , Spectrum Analysis/methods , Adenosine Triphosphate/biosynthesis , Animals , Carbon/metabolism , Culture Media , Energy Metabolism , Heart/physiology , Mitochondria/metabolism , Oxidation-Reduction , Oxygen Consumption , Perfusion , Rabbits , Reperfusion Injury , Reproducibility of ResultsABSTRACT
X-ray crystal structures of bc1 complexes obtained over the last 15 years have provided a firm structural basis for our understanding of the complex. For the most part there is good agreement between structures from different species, different crystal forms, and with different inhibitors bound. In this review we focus on some of the remaining unexplained differences, either between the structures themselves or the interpretations of the structural observations. These include the structural basis for the motion of the Rieske iron-sulfur protein in response to inhibitors, a possible conformational change involving tyrosine132 of cytochrome (cyt) b, the presence of cis-peptides at the beginnings of transmembrane helices C, E, and H, the structural insight into the function of the so-called "Core proteins", different modelings of the retained signal peptide, orientation of the low-potential heme b, and chirality of the Met ligand to heme c1. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.
Subject(s)
Electron Transport Complex III/chemistry , Protein Conformation , Amino Acid Sequence , Animals , Electron Transport Complex III/genetics , Electron Transport Complex III/metabolism , Heme/chemistry , Heme/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Sequence Homology, Amino AcidABSTRACT
The X-ray crystal structure of ribosome hibernation promoting factor (HPF) from Vibrio cholerae is presented at 2.0â Å resolution. The crystal was phased by two-wavelength MAD using cocrystallized cobalt. The asymmetric unit contained two molecules of HPF linked by four Co atoms. The metal-binding sites observed in the crystal are probably not related to biological function. The structure of HPF has a typical ß-α-ß-ß-ß-α fold consistent with previous structures of YfiA and HPF from Escherichia coli. Comparison of the new structure with that of HPF from E. coli bound to the Thermus thermophilus ribosome [Polikanov et al. (2012), Science, 336, 915-918] shows that no significant structural changes are induced in HPF by binding.
Subject(s)
Cobalt/chemistry , Escherichia coli Proteins/chemistry , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Vibrio cholerae/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Escherichia coli/chemistry , Escherichia coli/metabolism , Escherichia coli Proteins/isolation & purification , Models, Molecular , Molecular Sequence Data , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Ribosomal Proteins/isolation & purification , Ribosomes/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Thermus thermophilus/chemistry , Thermus thermophilus/metabolism , Vibrio cholerae/metabolismABSTRACT
Database analysis and molecular mechanics were used to determine the conformational flexibility of tridentate scorpionate ligands. The tris(pyrazolyl)methane and tris(pyrazolyl)borate ligands act like molecular vises, opening their tripodal structure for larger metals and closing around smaller metal ions. Tris(3-tert-butylpyrazolyl)methane has significant preference for larger metal ions than its unsubstituted parent compound. Tris(pyrazolyl)methanes and tris(pyrazolyl)borates have similar conformational flexibilities. Placing sterically hindered groups on the central carbon or boron has only a minor effect on the geometry of the tris(pyrazolyl)methanes and tris(pyrazolyl)borates. However, it does influence the flexibility of the ligands, particularly when they have to open far from their ideal geometry, which commonly occurs.
