Bernd Ludwig

Cytochrome c Oxidase and the Regulation of Oxidative Phosphorylation

Life of higher organisms is essentially dependent on the efficient synthesis of ATP by oxidative phosphorylation in mitochondria. An important and as yet unsolved question of energy metabolism is how are the variable rates of ATP synthesis at maximal work load during exercise or mental work and at rest or during sleep regulated. This article reviews our present knowledge on the structure of bacterial and eukaryotic cytochrome c oxidases and correlates it with recent results on the regulatory functions of nuclear‐coded subunits of the eukaryotic enzyme, which are absent from the bacterial enzyme. A new molecular hypothesis on the physiological regulation of oxidative phosphorylation is proposed, assuming a hormonally controlled dynamic equilibrium in vivo between two states of energy metabolism, a relaxed state with low ROS (reactive oxygen species) formation, and an excited state with elevated formation of ROS, which are known to accelerate aging and to cause degenerative diseases and cancer. The hypothesis is based on the allosteric ATP inhibition of cytochrome c oxidase at high intramitochondrial ATP/ADP ratios (“second mechanism of respiratory control”), which is switched on by cAMP‐dependent phosphorylation and switched off by calcium‐induced dephosphorylation of the enzyme.

Cytochrome c Oxidase (Heme aa3) from Paracoccus denitrificans: Analysis of Mutations in Putative Proton Channels of Subunit I

One of the challenging features of energy-transducing terminal oxidases, like the aa3 cytochrome c oxidase of Paracoccus denitrificans, is the translocation of protons across the cytoplasmic membrane, which is coupled to the transfer of electrons to oxygen. As a prerequisite for a more advanced examination of the enzymatic properties, several amino acid residues, selected on the basis of recent three-dimensional structure determinations, were exchanged in subunit I of the Paracoccus enzyme by site-directed mutagenesis. The properties of the mutated oxidases were analyzed by different methods to elucidate whether they are involved in the coupled and coordinated transfer of protons via two different pathways either to the site of oxygen reduction or through the enzyme from the cytoplasm to the periplasmic side.

The Rieske protein from Paracoccus denitrificans is inserted into the cytoplasmic membrane by the twin‐arginine translocase

The Rieske [2Fe−2S] protein (ISP) is an essential subunit of cytochrome bc1 complexes in mitochondrial and bacterial respiratory chains. Based on the presence of two consecutive arginines, it was argued that the ISP of Paracoccus denitrificans, a Gram‐negative soil bacterium, is inserted into the cytoplasmic membrane via the twin‐arginine translocation (Tat) pathway. Here, we provide experimental evidence that membrane integration of the bacterial ISP indeed relies on the Tat translocon. We show that targeting of the ISP depends on the twin‐arginine motif. A strict requirement is established particularly for the second arginine residue (R16); conservative replacement of the first arginine (R15K) still permits substantial ISP transport. Comparative sequence analysis reveals characteristics common to Tat signal peptides in several bacterial ISPs; however, there are distinctive features relating to the fact that the presumed ISP Tat signal simultaneously serves as a membrane anchor. These differences include an elevated hydrophobicity of the h‐region compared with generic Tat signals and the absence of an otherwise well‐conserved ‘+5’‐consensus motif lysine residue. Substitution of the +5 lysine (Y20K) compromises ISP export and/or cytochrome bc1 stability to some extent and points to a specific role for this deviation from the canonical Tat motif. EPR spectroscopy confirms cytosolic insertion of the [2Fe−2S] cofactor. Mutation of an essential cofactor binding residue (C152S) decreases the ISP membrane levels, possibly indicating that cofactor insertion is a prerequisite for efficient translocation along the Tat pathway.

Direct Demonstration of Half-of-the-sites Reactivity in the Dimeric Cytochrome bc1 Complex ENZYME WITH ONE INACTIVE MONOMER IS FULLY ACTIVE BUT UNABLE TO ACTIVATE THE SECOND UBIQUINOL OXIDATION SITE IN RESPONSE TO LIGAND BINDING AT THE UBIQUINONE REDUCTION SITE

We previously proposed that the dimeric cytochrome bc1 complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc1 monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289–22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc1 complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10–16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c1 by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wild-type dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.

The structure of porin from Paracoccus denitrificans at 3.1 A resolution

© 1997 Federation of European Biochemical Societies.

Surf1, Associated with Leigh Syndrome in Humans, Is a Heme-binding Protein in Bacterial Oxidase Biogenesis

Biogenesis of mitochondrial cytochrome c oxidase (COX) relies on a large number of assembly factors, among them the transmembrane protein Surf1. The loss of human Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder caused by severe COX deficiency. In the bacterium Paracoccus denitrificans, two homologous proteins, Surf1c and Surf1q, were identified, which we characterize in the present study. When coexpressed in Escherichia coli together with enzymes for heme a synthesis, the bacterial Surf1 proteins bind heme a in vivo. Using redox difference spectroscopy and isothermal titration calorimetry, the binding of the heme cofactor to purified apo-Surf1c and apo-Surf1q is quantified: Each of the Paracoccus proteins binds heme a in a 1:1 stoichiometry and with Kd values in the submicromolar range. In addition, we identify a conserved histidine as a residue crucial for heme binding. Contrary to most earlier concepts, these data support a direct role of Surf1 in heme a cofactor insertion into COX subunit I by providing a protein-bound heme a pool.

Heterologous expression of soluble fragments of cytochrome c552 acting as electron donor to the Paracoccus denitrificans cytochrome c oxidase

A membrane-bound c-type cytochrome, c552, acts as the electron mediator between the cytochrome bc1 complex and cytochrome c oxidase in the branched respiratory chain of the bacterium Paracoccus denitrificans. Unlike in mitochondria where a soluble cytochrome c interacts with both complexes, the bacterial c552, the product of the cycM gene, shows a tripartite structure, with an N-terminal membrane anchor separated from a typical class I cytochrome domain by a highly charged region. Two derivative fragments, lacking either only the membrane spanning region or both N-terminal domains, were constructed on the genetic level, and expressed in Escherichia coli cotransformed with the ccm gene cluster encoding host-specific cytochrome c maturation factors. High levels of cytochromes c were expressed and located in the periplasm as holo-proteins; both these purified c552 fragments are functional in electron transport to oxidase, as ascertained by kinetic measurements, and will prove useful for future structural studies of complex formation by NMR and X-ray diffraction.

Site-directed mutagenesis of cytochrome c oxidase reveals two acidic residues involved in the binding of cytochrome c

Site-directed mutagenesis in subunit II of the cytochrome c oxidase (haem aa3) from Paracoccus denitrificans reveals that two carboxylic residues, Glu-246 and Asp-206 (corresponding to 198 and 158 in the bovine subunit II), are involved in the binding of cytochrome c. Spectrophotometric and polarographic measurements with the isolated enzymes of both mutant strains show a strongly reduced activity compared to wild-type oxidase, with the overall catalytic capacity (kcat/KM) of both mutants decreased about 8-fold. EPR spectra reveal no significant differences between the wild-type and the mutant enzymes, indicating that neither residue contributes significantly to the structure of the CuA centre. We conclude that Glu-246 and Asp-206 constitute an essential part of the binding site for cytochrome c.

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.

Lipidomics of intact mitochondria by MALDI-TOF/MS

A simple and fast method of lipid analysis of isolated intact mitochondria by means of MALDI-TOF mass spectrometry is described. Mitochondria isolated from bovine heart and yeast have been employed to set up and validate the new method of lipid analysis. The mitochondrial suspension is directly applied over the target and, after drying, covered by a thin layer of the 9-aminoacridine matrix solution. The lipid profiles acquired with this procedure contain all peaks previously obtained by analyzing the lipid extracts of isolated mitochondria by TLC and/or mass spectrometry. The novel procedure allows the quick, simple, precise, and accurate analysis of membrane lipids, utilizing only a tiny amount of isolated organelle; it has also been tested with intact membranes of the bacterium Paracoccus denitrificans for its evolutionary link to present-day mitochondria. The method is of general validity for the lipid analysis of other cell fractions and isolated organelles.