W. John Ingledew

A re-evaluation of the spectral, potentiometric and energy-linked properties of cytochrome c oxidase in mitochondria.

In this paper we analyze recent results on the spectral, potentiometric and energy-linked properties of cytochrome c oxidase. It is demonstrated that the current hypothesis in which cytochromes a and a3 are assumed to contribute equally to the a-band at 605 nm disagrees with spectral information, and that this band is mostly, perhaps entirely, due to cytochrome a. The two heroes of cytochrome oxidase interact strongly, this interaction being reflected extensively in the redox properties of the heroes (midpoint redox potentials, Era) and also in the spin state and light absorption spectra. The effects of the ligands to cytochrome c oxidase are discussed in some detail in view of the new information. Special attention is given to the implications of the heine-berne interaction for the mechanism of action of the enzyme and its control by the mitochondrial energy state.

A potentiometric and kinetic study on the respiratory chain of ferrous-iron-grown Thiobacillus ferrooxidans.

The type and number of respiratory chain components present in membranes of Thiobacillus ferrooxidans have been investigated. These redox components were resolved potentiometrically and kinetically. Using optical techniques two cytochromes a1, multiple cytochromes c and two cytochromes b were detected. By using electron paramagnetic resonance, two copper-containing centres, high and low spin ferric haems and a ferredoxin centre were detected. Based on the combination of a potentiometric resolution and a kinetic study a model for the sequence of the respiratory chain components in the Fe2+ to O2 branch of the T. ferrooxidans respiratory chain is proposed.

Studies on electron paramagnetic resonance spectra manifested by a respiratory chain hydrogen carrier

Abstract The high-potential iron-sulfur protein (HiPIP) center of succinate dehydrogenase has an electron paramagnetic resonance (epr) signal in the oxidized form, centered at g = 2.01, and under certain conditions this epr signal is accompanied by absorbances at g = 2.04, g = 1.99, and g = 1.96. These absorbances have been attributed to a spin-spin interaction of paramagnetic species, the semiquinone form of ubiquinone being involved (Ruzicka et al., Proc. Nat. Acad. Sci. USA 72 , 2886). In the present work this magnetic interaction is studied further; it is concluded that of the three possible species (HiPIP, Flavin H and UQH (ubiquinone)) which may interact with UQH; a second UQ most likely partner for the interaction. Nonetheless, the HiPIP center of succinate dehydrogenase also plays a role in the interaction by acting as a “magnetic relaxer” of one or both of the interacting UQHs. The physiological reaction of that part of the ubiquinone pool associated with the succinate dehydrogenase (on the matrix side of the inner mitochondrial membrane) is UQH 2 ⇌ UQH + H + + e − . This is in line with recent postulates of the mechanism of ubiquinone mediation in electron transfer.

PELDOR Spectroscopy Distance Fingerprinting of the Octameric Outer‐Membrane Protein Wza from Escherichia coli.

Distance fingerprinting: Pulsed electron-electron double resonance spectroscopy (PELDOR) is applied to the octameric membrane protein complex Wza of E. coli. The data yielded a detailed distance fingerprint of its periplasmic region that compares favorably to the crystal structure. These results provide the foundation to study conformation changes from interaction with partner proteins.

A thermodynamic analysis of the plasma membrane electron transport components in photoheterotrophically grown cells of Chloroflexus aurantiacus: An optical and electron paramagnetic resonance study

The thermodynamic and spectroscopic properties of the membrane bound iron‐sulphur centers and cytochromes in phototrophically grown cells of the thermophilic facultative photosynthetic green bacterium Chloroftexus aurantiacus have been examined. The present study shows that Chloroflexus contains several cytochromes of b and c type, along with multiple ferredoxin‐like centers with resonances centered at g = 1.93, g = 2.017 (HiPIP‐like center) and g = 1.90 (Rieske‐type protein). The g = 1.90 centre appears to be actively involved in photooxidative reactions. These observations suggest the presence of a previously undetected cytochrome complex in this bacterium.

The purification of a respiratory oxidase complex from escherichia coli

has the ability to regulate the composition of its respiratory chain according to environmental factors [ 1,2]. When grown aerobically beyond exponential phase, cells produce two respira- tory oxidases, cytochrome o and cytochrome d [3]. When grown anaerobically with a non-fermentable carbon source (e.g., glycerol) plus fumarate, cells contain fumarate reductase as the major terminal respiratory enzyme. However these cells also contain very high concentrations of the oxidase cytochrome d, while no cytochrom& o is spectroscopically detect- able. Cytochromes

Energetic problems faced by micro-organisms growing or surviving on parsimonious energy sources and at acidic pH: I. Acidithiobacillus ferrooxidans as a paradigm.

The mitochondrial paradigm for a chemiosmotic energy transduction mechanism requires frequently misunderstood modifications for application to microbes growing and surviving at acidic pH values and/or with relatively weak reductants as energy sources. Here the bioenergetics of the iron oxidiser Acidithiobacillus ferrooxidans are reviewed and analysed so as to develop the general bioenergetic principles for understanding organisms that grow under these conditions. Extension of the principles outlined herein to organisms that survive (as opposed to grow) under these conditions is to be presented in a subsequent article.

EPR studies of higher plant mitochondria I. Ubisemiquinone and its relation to alternative respiratory oxidations

An EPR investigation of the region of the higher plant respiratory chain involving ubiquinone and Center S-3 of succinate dehydrogenase is reported. At temperatures close to those of liquid helium, first derivative spectra corresponding to Center S-3 (gmax = 2.017) and a signal split around g = 2.00 (major features of peaks and troughs at g values of 2.045, 2.03, 1.985, 1.97 and 1.96) were observed in mung bean (Phaseolus aureus), Arum maculatum spadix, Sauromatum guttatum spadix and tulip bulb (Tulipa gesnerana) mitochondria. The split signal was small or absent in potato tuber and Symplocarpus foetidus spadix mitochondria. The redox behavior of these signals in mung bean mitochondria in a variety of respiratory steady-state conditions suggested that the components giving rise to them were an integral part of the respiratory chain and were located on the substrate side of coupling Site II. The split signal could be removed by addition of hydroxamic acids in all tissues tested, although the Ks of this effect was an order of magnitude higher than the Ki of inhibition of the alternative respiratory pathway in mung bean and Sauromatum guttatum spadix mitochondria. The results are discussed in relation to the current ideas on the ordering of components in the region around the classical Site II of the respiratory chain and in relation to the location of the alternative respiratory oxidase pathway of higher plants.

Thermodynamic resolution of the iron-sulfur centers of the succinic dehydrogenase of Rhodopseudomonas sphaeroides

Abstract A single alkaline wash removes most of the succinic dehydrogenase activity from chromatophores of Rhodopseudomonas sphaeroides. Three iron-sulfur centers are also removed by this washing. Two of these are ferredoxin-like centers with electron paramagnetic resonance signals at gv = 1.94 and midpoint potentials of +50 and −250 mV at pH 7. The third is a high-potential iron-sulfur protein type signal centered at g 2.01 and a midpoint potential of +80 mV at pH 7. These centers have very similar properties to those of the well-characterized mammalian succinic dehydrogenase and account for the majority of iron-sulfur centers observed in chromatophores. Because it is so easily removed, it is concluded that succinic dehydrogenase is located on the outer surface of the chromatophore membrane, a conclusion supported by the fact that removal of the enzyme does not interfere with the kinetics of light-induced electron flow, nor does it allow cytochrome c2 to escape from inside the chromatophore vesicles.

Dimer:dimer stacking Interactions are important for nucleic acid binding by the archaeal chromatin protein Alba

Archaea use a variety of small basic proteins to package their DNA. One of the most widespread and highly conserved is the Alba (Sso10b) protein. Alba interacts with both DNA and RNA in vitro, and we show in the present study that it binds more tightly to dsDNA (double-stranded DNA) than to either ssDNA (single-stranded DNA) or RNA. The Alba protein is dimeric in solution, and forms distinct ordered complexes with DNA that have been visualized by electron microscopy studies; these studies suggest that, on binding dsDNA, the protein forms extended helical protein fibres. An end-to-end association of consecutive Alba dimers is suggested by the presence of a dimer–dimer interface in crystal structures of Alba from several species, and by the strong conservation of the interface residues, centred on Arg59 and Phe60. In the present study we map perturbation of the polypeptide backbone of Alba upon binding to DNA and RNA by NMR, and demonstrate the central role of Phe60 in forming the dimer–dimer interface. Site-directed spin labelling and pulsed ESR are used to confirm that an end-to-end, dimer–dimer interaction forms in the presence of dsDNA.