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.

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How mitochondria produce reactive oxygen species.

Infrared spectroscopy of proteins.

O2- oxidizes the [4Fe-4S] clusters of dehydratases, such as aconitase, causing-inactivation and release of Fe(II), which may then reduce H2O2 to OH- +OH.. SODs inhibit such HO. production by scavengingO2-, but Cu, ZnSODs, by virtue of a nonspecific peroxidase activity, may peroxidize spin trapping agents and thus give the appearance of catalyzing OH. production from H2O2. There is a glycosylated, tetrameric Cu, ZnSOD in the extracellular space that binds to acidic glycosamino-glycans. It minimizes the reaction of O2- with NO. E. coli, and other gram negative microorganisms, contain a periplasmic Cu, ZnSOD that may serve to protect against extracellular O2-. Mn(III) complexes of multidentate macrocyclic nitrogenous ligands catalyze the dismutation of O2- and are being explored as potential pharmaceutical agents. SOD-null mutants have been prepared to reveal the biological effects of O2-. SodA, sodB E. coli exhibit dioxygen-dependent auxotrophies and enhanced mutagenesis, reflecting O2(-)-sensitive biosynthetic pathways and DNA damage. Yeast, lacking either Cu, ZnSOD or MnSOD, are oxygen intolerant, and the double mutant was hypermutable and defective in sporulation and exhibited requirements for methionine and lysine. A Cu, ZnSOD-null Drosophila exhibited a shortened lifespan.

Superoxide Radical and Superoxide Dismutases

High-throughput techniques are leading to an explosive growth in the size of biological databases and creating the opportunity to revolutionize our understanding of life and disease Interpretation of these data remains, however, a major scientific challenge Here, we propose a methodology that enables us to extract and display information contained in complex networks1,2,3 Specifically, we demonstrate that we can find functional modules4,5 in complex networks, and classify nodes into universal roles according to their pattern of intra- and inter-module connections The method thus yields a ‘cartographic representation’ of complex networks Metabolic networks6,7,8 are among the most challenging biological networks and, arguably, the ones with most potential for immediate applicability9 We use our method to analyse the metabolic networks of twelve organisms from three different superkingdoms We find that, typically, 80% of the nodes are only connected to other nodes within their respective modules, and that nodes with different roles are affected by different evolutionary constraints and pressures Remarkably, we find that metabolites that participate in only a few reactions but that connect different modules are more conserved than hubs whose links are mostly within a single module

Functional cartography of complex metabolic networks

Copper is an essential trace element in living systems, present in the parts per million concentration range. It is a key cofactor in a diverse array of biological oxidation-reduction reactions. These involve either outer-sphere electron transfer, as in the blue copper proteins and the Cu{sub A} site of cytochrome oxidase and nitrous oxide redutase, or inner-sphere electron transfer in the binding, activation, and reduction of dioxygen, superoxide, nitrite, and nitrous oxide. Copper sites have historically been divided into three classes based on their spectroscopic features, which reflect the geometric and electronic structure of the active site: type 1 (T1) or blue copper, type 2 (T2) or normal copper, and type 3 (T3) or coupled binuclear copper centers. 428 refs.

Multicopper Oxidases and Oxygenases

Keilin9s classic paper of 1925 [Keilin (1925) Proc. R. Soc. London Ser. B 100 , 129–151], achieved with simple, but elegant, techniques, describes the cytochrome components of the respiratory chain and their roles in intracellular respiration and oxygen consumption. Since that time, a tremendous amount of work has clarified the intricate details of the prosthetic groups, cofactors and proteins that comprise the respiratory chain and associated machinery for ATP synthesis. The work has culminated in advanced crystallographic and spectroscopic methods that provide structural and mechanistic details of this mitochondrial molecular machinery, in many instances to atomic level. I review here the current state of understanding of the mitochondrial respiratory chain in terms of structures and dynamics of the component proteins and their roles in the biological electron and proton transfer processes that result in ATP synthesis. These advances, together with emerging evidence of further diverse roles of mitochondria in health and disease, have prompted a new era of interest in mitochondrial function.

The molecular machinery of Keilin's respiratory chain.

Electron and proton transfers through quinones and cytochrome bc complexes

Redox-controlled phosphorylation of thylakoid membrane proteins represents a unique system for the regulation of light energy utilization in photosynthesis The molecular mechanisms for this process remain unknown, but current views suggest that the plastoquinone pool directly controls the activation of the kinase On the basis of enzyme activation by a pH shift in the darkness combined with flash photolysis, EPR, and optical spectroscopy we propose that activation occurs when plastoquinol occupies the quinol-oxidation (Qo) site of the cytochrome bf complex, having its high-potential path components in a reduced state A linear correlation between kinase activation and accessibility of the Qo site to plastoquinol was established by quantification of the shift in the gy EPR signal of the Rieske Fe–S center resulting from displacement of the Qo-site plastoquinol by a quinone analog Activity persists as long as one plastoquinol per cytochrome bf is still available Withdrawal of one electron from this plastoquinol after a single-turnover flash exciting photosystem I leads to deactivation of the kinase parallel with a decrease in the gz EPR signal of the reduced Rieske Fe–S center Cytochrome f, plastocyanin, and P700 are rereduced after the flash, indicating that the plastoquinol at the Qo site is limiting in maintaining the kinase activity These results give direct evidence for a functional cytochrome bf–kinase interaction, analogous to a signal transduction system where the cytochrome bf is the receptor and the ligand is the plastoquinol at the Qo site

https://www.jstor.org/stable/pdfplus/41375.pdf

Plastoquinol at the quinol oxidation site of reduced cytochrome bf mediates signal transduction between light and protein phosphorylation : thylakoid protein kinase deactivation by a single-turnover flash

Peter R. Rich

Papers

The molecular machinery of Keilin's respiratory chain.

Electron and proton transfers through quinones and cytochrome bc complexes

Plastoquinol at the quinol oxidation site of reduced cytochrome bf mediates signal transduction between light and protein phosphorylation : thylakoid protein kinase deactivation by a single-turnover flash

A motif for quinone binding sites in respiratory and photosynthetic systems.

The sites of superoxide anion generation in higher plant mitochondria.