The production of ROS (reactive oxygen species) by mammalian mitochondria is important because it underlies oxidative damage in many pathologies and contributes to retrograde redox signalling from the organelle to the cytosol and nucleus. Superoxide (O2•−) is the proximal mitochondrial ROS, and in the present review I outline the principles that govern O2•− production within the matrix of mammalian mitochondria. The flux of O2•− is related to the concentration of potential electron donors, the local concentration of O2 and the second-order rate constants for the reactions between them. Two modes of operation by isolated mitochondria result in significant O2•− production, predominantly from complex I: (i) when the mitochondria are not making ATP and consequently have a high Δp (protonmotive force) and a reduced CoQ (coenzyme Q) pool; and (ii) when there is a high NADH/NAD+ ratio in the mitochondrial matrix. For mitochondria that are actively making ATP, and consequently have a lower Δp and NADH/NAD+ ratio, the extent of O2•− production is far lower. The generation of O2•− within the mitochondrial matrix depends critically on Δp, the NADH/NAD+ and CoQH2/CoQ ratios and the local O2 concentration, which are all highly variable and difficult to measure in vivo. Consequently, it is not possible to estimate O2•− generation by mitochondria in vivo from O2•−-production rates by isolated mitochondria, and such extrapolations in the literature are misleading. Even so, the description outlined here facilitates the understanding of factors that favour mitochondrial ROS production. There is a clear need to develop better methods to measure mitochondrial O2•− and H2O2 formation in vivo, as uncertainty about these values hampers studies on the role of mitochondrial ROS in pathological oxidative damage and redox signalling.

/pdf/how-mitochondria-produce-reactive-oxygen-species-4spvs9r9mw.pdf

How mitochondria produce reactive oxygen species.

Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.

For present purposes, a protein-bound metal site consists of one or more metal ions and all protein side chain and exogenous bridging and terminal ligands that define the first coordination sphere of each metal ion. Such sites can be classified into five basic types with the indicated functions: (1) structural -- configuration (in part) of protein tertiary and/or quaternary structure; (2) storage -- uptake, binding, and release of metals in soluble form: (3) electron transfer -- uptake, release, and storage of electrons; (4) dioxygen binding -- metal-O{sub 2} coordination and decoordination; and (5) catalytic -- substrate binding, activation, and turnover. The authors present here a classification and structure/function analysis of native metal sites based on these functions, where 5 is an extensive class subdivided by the type of reaction catalyzed. Within this purview, coverage of the various site types is extensive, but not exhaustive. The purpose of this exposition is to present examples of all types of sites and to relate, insofar as is currently feasible, the structure and function of selected types. The authors largely confine their considerations to the sites themselves, with due recognition that these site features are coupled to protein structure at all levels. In themore » next section, the coordination chemistry of metalloprotein sites and the unique properties of a protein as a ligand are briefly summarized. Structure/function relationships are systematically explored and tabulations of structurally defined sites presented. Finally, future directions in bioinorganic research in the context of metal site chemistry are considered. 620 refs.« less

/pdf/structural-and-functional-aspects-of-metal-sites-in-biology-a6d65lev8n.pdf

Structural and Functional Aspects of Metal Sites in Biology

▪ Abstract Stably folded membrane proteins reside in a free energy minimum determined by the interactions of the peptide chains with each other, the lipid bilayer hydrocarbon core, the bilayer interface, and with water. The prediction of three-dimensional structure from sequence requires a detailed understanding of these interactions. Progress toward this objective is summarized in this review by means of a thermodynamic framework for describing membrane protein folding and stability. The framework includes a coherent thermodynamic formalism for determining and describing the energetics of peptide-bilayer interactions and a review of the properties of the environment of membrane proteins—the bilayer milieu. Using a four-step thermodynamic cycle as a guide, advances in three main aspects of membrane protein folding energetics are discussed: protein binding and folding in bilayer interfaces, transmembrane helix insertion, and helix-helix interactions. The concepts of membrane protein stability that emerge p...

/pdf/membrane-protein-folding-and-stability-physical-principles-2i9emery3i.pdf

MEMBRANE PROTEIN FOLDING AND STABILITY: Physical Principles

Topology of Superoxide Production from Different Sites in the Mitochondrial Electron Transport Chain

Mitochondrial cytochrome bc1 complex performs two functions: It is a respiratory multienzyme complex and it recognizes a mitochondrial targeting presequence. Refined crystal structures of the 11-subunit bc1 complex from bovine heart reveal full views of this bifunctional enzyme. The "Rieske" iron-sulfur protein subunit shows significant conformational changes in different crystal forms, suggesting a new electron transport mechanism of the enzyme. The mitochondrial targeting presequence of the "Rieske" protein (subunit 9) is lodged between the two "core" subunits at the matrix side of the complex. These "core" subunits are related to the matrix processing peptidase, and the structure unveils how mitochondrial targeting presequences are recognized.

https://science.sciencemag.org/content/sci/281/5373/64.full.pdf

Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex.

Structure of a water soluble fragment of the ‘Rieske’ iron–sulfur protein of the bovine heart mitochondrial cytochrome bc1 complex determined by MAD phasing at 1.5 å resolution

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2887%2981546-6

Solute carriers involved in energy transfer of mitochondria form a homologous protein family.

Zinc Ions Inhibit the Qp Center of Bovine Heart Mitochondrial bc1 Complex by Blocking a Protonatable Group

The role of the 'rieske' iron sulfur protein in the hydroquinone oxidation(qp) site of the cytochrome bc1 complex : the 'proton-gated affinity change ' mechanism

Thomas A. Link

Papers

Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex.

Structure of a water soluble fragment of the ‘Rieske’ iron–sulfur protein of the bovine heart mitochondrial cytochrome bc1 complex determined by MAD phasing at 1.5 å resolution

Solute carriers involved in energy transfer of mitochondria form a homologous protein family.

Zinc Ions Inhibit the Qp Center of Bovine Heart Mitochondrial bc1 Complex by Blocking a Protonatable Group

The role of the 'rieske' iron sulfur protein in the hydroquinone oxidation(qp) site of the cytochrome bc1 complex : the 'proton-gated affinity change ' mechanism