Gerhard Buse

Inhibition of cytochrome c oxidase function by dicyclohexylcarbodiimide

Dicyclohexylcarbodiimide (DCCD) reacted with beef heart cytochrome c oxidase in inhibit the proton-pumping function of this enzyme and to a lesser extent to inhibit electron transfer. The modification of cytochrome c oxidase in detergent dispersion or in vesicular membranes was in subunits II-IV. Labelling followed by fragmentation studies showed that there is one major site of modification in subunit III. DCCD was also incorporated into several sites in subunit II and at least one site of subunit IV. The major site in subunit III has a specificity for DCCD at least one order of magnitude greater than that of other sites (in subunits II and IV). Its modification could account for all of the observed effects of the reagent, at least for low concentrations of DCCD. Labelling of subunit II by DCCD was blocked by prior covalent attachment of arylazidocytochrome c, a cytochrome c derivative which binds to the high-affinity binding site for the substrate. The major site of DCCD binding in subunit III was sequenced. The label was found in glutamic acid 90 which is in a sequence of eight amino acids remarkably similar to the DCCD-binding site within the proteolipid protein of the mitochondrial ATP synthetase.

Electrical current generation and proton pumping catalyzed by the ba3-type cytochrome c oxidase from Thermus thermophilus

Several amino acid residues that have been shown to be essential for proton transfer in most cytochrome c oxidases are not conserved in the ba 3‐type cytochrome c oxidase from the thermophilic eubacterium Thermus thermophilus. So far, it has been unclear whether the Th. thermophilus ba 3‐type cytochrome c oxidase can nevertheless function as an electrogenic proton pump. In this study, we have combined charge translocation measurements on a lipid bilayer with two independent methods of proton pumping measurements to show that enzymatic turnover of the Th. thermophilus cytochrome c oxidase is indeed coupled to the generation of an electrocurrent and proton pumping across the membrane. In addition to a ‘vectorial’ consumption of 1.0 H+/e− for water formation, proton pumping with a stoichiometry of 0.4–0.5 H+/e− was observed. The implications of these findings for the mechanism of redox‐coupled proton transfer in this unusual cytochrome c oxidase are discussed.

Tissue‐specific genes code for polypeptide VIa of bovine liver and heart cytochrome c oxidase

Cytochrome c oxidase from higher eucaryotes is composed of 12-13 different protein components (review [1-3]). This was concluded from: (i) The occurrence of 12 protein bands after SDS gel electrophoresis in enzyme preparations from many species and tissues [4,5 ]; (ii) The isolation of 12 different polypeptides from bovine heart cytochrome c oxidase by gel chro- matography, and the proof of 12 different N-ter- minal amino acid sequences [6]; (iii) The occurrence of all components in the complex in stoichiometric amounts [4,7]; (iv) The immunoprecipitation of 12 polypeptides from a mitochondrial Triton X-100 lysate with a specific antiserum against subunit IV [8]. Recent work suggests that the functional properties of the enzyme are connected with the 3 large mito- chondriaUy synthesized subunits. The 2 heme a groups are assumed to be localized in subunits I and II, the 2 copper atoms in subunit II, which also binds cyto- chrome c, and the proton-pumping activity within subunit III (review [2,3]). The function of the other, cytoplasmically synthesized polypeptides remained unknown. Comparison of the gel electrophoretic polypeptide pattern of cytochrome c oxidases from rat liver and heart revealed differences in the apparent Mr-values of polypeptides Via [9]. (The nomenclature of [4] is used, if not otherwise stated.) Similar differences were found for polypeptide VIII of liver and heart cytochrome c oxidases from chicken, pig and bovine and for polypeptide VIIa from pig and bovine [5 ]. In a kinetic study of cytochrome c oxidases from bovine liver and heart, differences in the Vma x Kin-values for the 2 enzymes were observed [5,10]. It was concluded that vertebrates contain tissue-specific isoenzymes of cytochrome c oxidase. Here, the N-terminal amino acid sequences of cyto- chrome c oxidase polypeptides Via from bovine liver and bovine heart are compared. The data indicate that 2 different genes code for polypeptide Via of the enzymes from liver and heart.

Mapping of the cytochrome c binding site on cytochrome c oxidase

Cytochrome c oxidase (EC 1.9.3.1) is the terminal enzyme of the mitochondria respiratory chain catalysing electron transfer from cytochrome c to molecular oxygen [ 1,2]. The molecular mechanism of this process is still not understood. At present, little is known about such important structural features as the position of the prosthetic groups or the location and characteristics of the cytochrome c binding sites in the cytochrome c oxidase complex 131. The aim of this paper is to provide information about this latter problem as part of a more general effort of our laboratories to elucidate the relationship between structure and functions of cytochrome c oxidase. as in [S]. Cytochrome c oxidase was isolated from beef heart as in [7], the final dialysis step was omitted. The activity, measured polarographically in 0.5% Tween 80, 50 mM phosphate buffer (pH_7i4) ranged_fym 130170 mol cytochrome c. s . molaa3 . The covalent enzyme-substrate complex was

Sequence homology of bacterial and mitochondrial cytochrome c oxidases: Partial sequence data of cytochrome c oxidase from Paracoccus denitrificans

The aerobic electron transport chain of Paracoccus denitrificans is very similar to that of mitochondria. It has therefore been suggested that this bacterium might be evolutionarily related to mitochondria. The two subunits (Mr 45.000 and 28.000) of the Paracoccus cytochrome c oxidase were isolated and partially sequenced. The sequences were found to be surprisingly homologous to sequences of the subunits I and II of mitochondrial cytochrome c oxidases. The data provide a molecular basis for the symbiotic origin of mitochondria and strongly support the notion that in eucaryotic oxidases subunits I and/or II carry the redox centers, heme and copper.

The protein formula of beef heart cytochrome c oxidase.

Beef heart cytochrome c oxidase consist of 12 different polypeptides stoichiometrically arranged in respiratory complex IV. The functional 2 heme a, 2 copper monomer of this complex consist of 1793 amino acids; the exact Mr is 202,787 Da. From 17 cysteine residues, six are involved in the formation of three disulphide bonds. The theoretical heme a content of the enzyme is 9.86 nmol/mg protein. The theoretical iron and copper contents are 0.55 and 0.63 microgram/mg protein, respectively.

Membrane proteins: analysis of molecular and supramolecular structure: Workshop organized by the Sonderforschungsbereich 160 - Eigenschaften biologischer Membranen - October 5–7, 1980, Maria Laach, FRG

The Sonderforschungsbereich 160 organized this workshop to discuss and evaluate recent activities and trends in the field of membrane protein structure. Some 20 scientists were invited to present their most recent investigations in an attempt to shed some light on various facets of the structural organization of this rather elusive class of proteins. Despite all efforts there is so far not a single example where the structural determination of a membrane protein has reached the degree of comprehensiveness, accuracy and definiteness which we are eventually seeking in order to begin inquiring sensibly into the way these molecules operate. It was the central aim of the workshop to discuss how the various rapidly advancing physical and chemical approaches presently available can be combined to achieve a ‘holistic’ picture of the structure of a membrane protein. Ultimately it is only the atomic model which can provide us with the necessary structural information. Most advanced in this respect is bacteriorhodopsin. A model of this protein, obtained by piecing together information from various sources [ 11, was presented by R. Henderson (Cambridge). Low-dose electron microscopy combined with digital image reconstruction techniques provided the basic morphological information: The molecule is composed of seven helical rods traversing the membrane almost perpendicularly [2]. Analysis of a new two-dimensional crystal form obtained by recrystallisation [3] corroborated the boundaries of individual bacteriorhodopsin molecules in the lattice. The amino acid sequence [4] was then fitted tentatively into the morphological model. As important preliminaries the 7 helical segments and the link regions in the sequence were localized by applying the criteria of hydrophobicity and inaccessibility to proteolytic cleavage and assuming that the helices should be comparable in length. In the next step these helical segments had to be identified in the density map. To reduce the enormous number of possible assignments the following criteria were applied: (a) connectivity of non-helical link regions; (b) charge neutralization; (c) correlation of calculated electron scattering cross-sections for the individual helices with the densities determined experimentally. The model emerging as the most probable one exhibits a number of features favourable for a membrane-embedded proton pump. The buried charged amino acids are localized in the center of the molecule, possibly forming a hydrophilic channel through which protons could jump, whereas the hydrophobic, uncharged residues are directed outward towards the lipid environment. This ‘inside-out’ arrangement has meanwhile been confirmed by neutron diffraction experiments [5]. Further experimental verification of this model might be achieved either by electron optical means with two-dimensional crystals, pushing resolution further towards the instrumental limits, or by continuing efforts to prepare three-dimensional crystals suitable for X-ray analysis. Here considerable progress has been made recently, not only with bacteriorhodopsin [6], disproving the long prevailing opinion that crystallizing intrinsic membrane proteins is impossible. J. P. Rosenbusch (Basel) reported about successful attempts to obtain three-dimensional crystals of porin, a pore-forming transmembrane protein from the outer membrane of Escherichiu coli [7]. Several crystal

Respiratory Complex IV and Cytochrome a,a3

If one asks, which criteria might be mandatory for the opinion on an enzyme complex, the function of which is not yet fully understood, the integral stoichiometry of all components of the isolate must be considered a guideline for preparation and purification besides known enzymatic activities and pertinent spectral properties. Application of this principal chemical rule is especially necessary in those fields of work, where our methods leave doubt, whether what we obtain is a faithful representation of what exists in nature. This is the case with the respiratory enzyme complexes, e.g. mitochondrial cytochrome c oxidase. The solubilisation and purification of this enzyme from the mitochondrial (or bacterial) membrane has in many cases not met the above criterion. The complete proteinchemical description of the so far most complex type of this enzyme, obtained from bovine heart, and its comparison with simpler eucaryotic and procaryotic oxidases defines the structure of integral preparations more precisely and at the same time is a guide to the function.

Electrophoretically monodisperse cytochrome c oxidases

A discontinuous gradient polyacrylamide gel electrophoresis under nondenaturing conditions has been used to demonstrate monodispersity of procaryotic and eucaryotic cytochrome c oxidase preparations. Alkaline treated bovine enzyme which contains nine subunits as analysed by subsequent discontinuous SDS-polyacrylamide gel electrophoresis is a monodisperse dimer in 0.1% Triton X-100 and a monomer in 0.1% dodecyl maltoside. The Mr-values corrected for bound detergent are 286,000 in Triton X-100 and 152,000 in dodecyl maltoside respectively. The two-subunit bacterial cytochrome c oxidase of Paracoccus denitrificans is proved to be a monomer with a corrected Mr of 76,000 in both nonionic detergents Triton X-100 and dodecyl maltoside.

The mechanism of the electron transfer process from cytochrome c to cytochrome c oxidase studied by resonance Raman spectroscopic techniques

In the terminal step of the respiratory chain of aerobic organisms, the membrane-bound enzyme cytochrome c oxidase (CcO) reduces dioxygen to water [1]. The required electrons are delivered stepwise by the soluble electron carrier cytochrome c (Cyt) [2]. Prior to these interprotein electron transfer reactions, Cyt binds to CcO via electrostatic interactions between the positively charged lysine-rich region on the surface of Cyt and a negatively charged binding domain of CcO [3]. Although the crystal structures of the individual redox proteins have been determined (e. g., [4, 5]), the structure of the Cyt-CcO protein complex is not known. On the other hand, a variety of spectroscopic results indicate that conformational changes are induced in the active sites of both partner proteins upon complex formation [6 – 8]. In our previous resonance Raman (RR) studies [7, 8], we could demonstrate that upon formation of the fully oxidized Cyt-CcO complex, a conformational equilibrium of the bound Cyt is established which involves two conformers, B1 and B2. These species are also formed upon binding to negatively charged model systems (electrodes, phospholipid vesicles, polyanions) which mimic the interaction domain of CcO [9, 10]. In contrast to Bl, which can be regarded as essentially identical to the unbound Cyt, the spectrum of state B2 reveals significant structural changes in the heme pocket of this conformer which most likely include the exchange of the Met-80 axial ligand presumably by a hydroxide [11] and are accompanied by a drastic lowering of the reduction potential [9]. These findings have been suggested to be of functional relevance for interprotein electron transfer to CcO [7, 8].