Elisabeth Darrouzet

Resistance mutations reveal the atovaquone‐binding domain of cytochrome b in malaria parasites

Atovaquone represents a class of antimicrobial agents with a broad‐spectrum activity against various parasitic infections, including malaria, toxoplasmosis and Pneumocystis pneumonia. In malaria parasites, atovaquone inhibits mitochondrial electron transport at the level of the cytochrome bc1 complex and collapses mitochondrial membrane potential. In addition, this drug is unique in being selectively toxic to parasite mitochondria without affecting the host mitochondrial functions. A better understanding of the structural basis for the selective toxicity of atovaquone could help in designing drugs against infections caused by mitochondria‐containing parasites. To that end, we derived nine independent atovaquone‐resistant malaria parasite lines by suboptimal treatment of mice infected with Plasmodium yoelii; these mutants exhibited resistance to atovaquone‐mediated collapse of mitochondrial membrane potential as well as inhibition of electron transport. The mutants were also resistant to the synergistic effects of atovaquone/ proguanil combination. Sequencing of the mitochondrially encoded cytochrome b gene placed these mutants into four categories, three with single amino acid changes and one with two adjacent amino acid changes. Of the 12 nucleotide changes seen in the nine independently derived mutants 11 replaced A:T basepairs with G:C basepairs, possibly because of reactive oxygen species resulting from atovaquone treatment. Visualization of the resistance‐conferring amino acid positions on the recently solved crystal structure of the vertebrate cytochrome bc1 complex revealed a discrete cavity in which subtle variations in hydrophobicity and volume of the amino acid side‐chains may determine atovaquone‐binding affinity, and thereby selective toxicity. These structural insights may prove useful in designing agents that selectively affect cytochrome bc1 functions in a wide range of eukaryotic pathogens.

Large scale domain movement in cytochrome bc1: a new device for electron transfer in proteins

Recently, crystallographic, spectroscopic, kinetic and biochemical genetic data have merged to unveil a large domain movement for the Fe-S subunit in cytochrome bc(1). In this evolutionarily conserved enzyme, the domain motion acts to conduct intra-complex electron transfer and is essential for redox energy conversion.

The 49‐kDa subunit of NADH‐ubiquinone oxidoreductase (Complex I) is involved in the binding of piericidin and rotenone, two quinone‐related inhibitors

Piericidin is a potent inhibitor of the mitochondrial and bacterial type I NADH‐ubiquinone oxidoreductases (Complex I) and is considered to bind at or close to the ubiquinone binding site(s) of the enzyme. Piericidin‐resistant mutants of the bacterium Rhodobacter capsulatus have been isolated and the present work demonstrates that a single missense mutation at the level of the gene encoding the peripheral 49‐kDa/NUOD subunit of Complex I is definitely associated with this resistance. Based on this original observation, we propose a model locating the binding site for piericidin (and quinone) at the interface between the hydrophilic and hydrophobic domains of Complex I.

The Complex I from Rhodobacter capsulatus

The NADH-ubiquinone oxidoreductase (type I NDH) of Rhodobacter capsulatus is a multisubunit enzyme encoded by the 14 genes of the nuo operon. This bacterial enzyme constitutes a valuable model for the characterization of the mitochondrial Complex I structure and enzymatic mechanism for the following reasons. (i) The mitochondria-encoded ND subunits are not readily accessible to genetic manipulation. In contrast, the equivalents of the mitochondrial ND1, ND2, ND4, ND4L, ND5 and ND6 genes can be easily mutated in R. capsulatus by homologous recombination. (ii) As illustrated in the case of ND1 gene, point mutations associated with human cytopathies can be reproduced and studied in this model system. (iii) The R. capsulatus model also allows the recombinant manipulations of iron-sulfur (Fe-S) subunits and the assignment of Fe-S clusters as illustrated in the case of the NUOI subunit (the equivalent of the mitochondrial TYKY subunit). (iv) Finally, like mitochondrial Complex I, the NADH-ubiquinone oxidoreductase of R. capsulatus is highly sensitive to the inhibitor piericidin-A which is considered to bind to or close to the quinone binding site(s) of Complex I. Therefore, isolation of R. capsulatus mutants resistant to piericidin-A represents a straightforward way to map the inhibitor binding sites and to try and define the location of quinone binding site(s) in the enzyme. These illustrations that describe the interest in the R. capsulatus NADH-ubiquinone oxidoreductase model for the general study of Complex I will be critically developed in the present review.

Inter-monomer Electron Transfer Between the Low Potential b Hemes of Cytochrome bc1

Cytochrome (cyt) bc(1) is a structural dimer with its monomers consisting of the Fe-S protein, cyt b, and cyt c(1) subunits. Its three-dimensional architecture depicts it as a symmetrical homodimer, but the mobility of the head domain of the Fe-S protein indicates that the functional enzyme exists in asymmetrical heterodimeric conformations. Here, we report a new genetic system for studying intra- and intermonomer interactions within the cyt bc(1) using the facultative phototrophic bacterium Rhodobacter capsulatus. The system involves two different sets of independently expressed cyt bc(1) structural genes carried by two plasmids that are coharbored by a cell without its endogenous enzyme. Our results indicate that coexpressed cyt bc(1) subunits were matured, assorted, and assembled in vivo into homo- and heterodimeric enzymes that can bear different mutations in each monomer. Using the system, the occurrence of intermonomer electron transfer between the low-potential b hemes of cyt bc(1) was probed by choosing mutations that perturb electron transfer at the hydroquinone oxidation (Q(o)) and quinone reduction (Q(i)) sites of the enzyme. The data demonstrate that active heterodimeric variants, formed of monomers carrying mutations that abolish only one of the two (Q(o) or Q(i)) active sites of each monomer, are produced, and they support photosynthetic growth of R. capsulatus. Detailed analyses of the physicochemical properties of membranes of these mutants, as well as purified homo- and heterodimeric cyt bc(1) preparations, demonstrated that efficient and productive electron transfer occurs between the low-potential b(L) hemes of the monomers in a heterodimeric enzyme. Overall findings are discussed with respect to intra- and intermonomer interactions that take place during the catalytic turnover of cyt bc(1).

The Cytochrome bc1 Complex and its Homologue the b6f Complex: Similarities and Differences

The ubihydroquinone:cytochrome c oxidoreductase (also called complex III, or bc1 complex), is a multi subunit enzyme encountered in a very broad variety of organisms including uni- and multi-cellular eukaryotes, plants (in their mitochondria) and bacteria. Most bacteria and mitochondria harbor various forms of the bc1 complex, while plant and algal chloroplasts as well as cyanobacteria contain a homologous protein complex called plastohydroquinone:plastocyanin oxidoreductase or b6f complex. Together, these enzyme complexes constitute the superfamily of the bc complexes. Depending on the physiology of the organisms, they often play critical roles in respiratory and photosynthetic electron transfer events, and always contribute to the generation of the proton motive force subsequently used by the ATP synthase. Primarily, this review is focused on comparing the ‘mitochondrial-type’ bc1 complex and the ‘chloroplast-type’ b6f complex both in terms of structure and function. Specifically, subunit composition, cofactor content and assembly, inhibitor sensitivity, proton pumping, concerted electron transfer and Fe—S subunit large-scale domain movement of these complexes are discussed. This is a timely undertaking in light of the structural information that is emerging for the b6f complex.

Revisiting Iodination Sites in Thyroglobulin with an Organ-oriented Shotgun Strategy

Thyroglobulin (Tg) is secreted by thyroid epithelial cells. It is essential for thyroid hormonogenesis and iodine storage. Although studied for many years, only indirect and partial surveys of its post-translational modifications were reported. Here, we present a direct proteomic approach, used to study the degree of iodination of mouse Tg without any preliminary purification. A comprehensive coverage of Tg was obtained using a combination of different proteases, MS/MS fragmentation procedures with inclusion lists and a hybrid mass high-resolution LTQ-Orbitrap XL mass spectrometer. Although only 16 iodinated sites are currently known for human Tg, we uncovered 37 iodinated tyrosine residues, most of them being mono- or diiodinated. We report the specific isotopic pattern of thyroxine modification, not recognized as a normal peptide pattern. Four hormonogenic sites were detected. Two donor sites were identified through the detection of a pyruvic acid residue in place of the initial tyrosine. Evidence for polypeptide cleavages sites due to the action of cathepsins and dipeptidyl proteases in the thyroid were also detected. This work shows that semi-quantitation of Tg iodination states is feasible for human biopsies and should be of significant medical interest for further characterization of human thyroid pathologies.

Genetic evidence for the existence of two quinone related inhibitor binding sites in NADH-CoQ reductase

Using the NADH-CoQ reductase of Rhodobacter capsulatus as a model for the mitochondrial Complex I, we have for the first time isolated bacterial mutants resistant to piericidin-A, a classical inhibitor of the mitochondrial enzyme. Their sensitivity to other inhibitors directed towards the quinone binding domain of complex I gives direct genetic evidence for the existence of two inhibitor binding sites.

Structure and Function of the Bacterial bc1 Complex: Domain Movement, Subunit Interactions, and Emerging Rationale Engineering Attempts

The ubiquinol: cytochrome c oxidoreductase, or the bc1 complex, is a key component ofboth respiratory and photosynthetic electron transfer and contributes to the formation of anelectrochemical gradient necessary for ATP synthesis. Numerous bacteria harbor a bc1 complexcomprised of three redox-active subunits, which bear two b-type hemes, one c-type heme, andone [2Fe–2S] cluster as prosthetic groups. Photosynthetic bacteria like Rhodobacter speciesprovide powerful models for studying the function and structure of this enzyme and are beingwidely used. In recent years, extensive use of spontaneous and site-directed mutants and theirrevertants, new inhibitors, discovery of natural variants of this enzyme in various species, andengineering of novel bc1 complexes in species amenable to genetic manipulations have providedus with a wealth of information on the mechanism of function, nature of subunit interactions,and assembly of this important enzyme. The recent resolution of the structure of variousmitochondrial bc1 complexes in different crystallographic forms has consolidated previousfindings, added atomic-scale precision to our knowledge, and raised new issues, such as thepossible movement of the Rieske Fe–S protein subunit during Qo site catalysis. Here, studiesperformed during the last few years using bacterial bc1 complexes are reviewed briefly andongoing investigations and future challenges of this exciting field are mentioned.

Distal genes of the nuo operon of Rhodobacter capsulatus equivalent to the mitochondrial ND subunits are all essential for the biogenesis of the respiratory NADH–ubiquinone oxidoreductase

Seven out of the 13 proteins encoded by the mitochondrial genome of mammals (peptides ND1 to ND6 plus ND4L) are subunits of the respiratory NADH–ubiquinone oxidoreductase (complex I). The function of these ND subunits is still poorly understood. We have used the NADH–ubiquinone oxidoreductase of Rhodobacter capsulatus as a model for the study of the function of these proteins. In this bacterium, the 14 genes encoding the NADH–ubiquinone oxidoreductase are clustered in the nuo operon. We report here on the biochemical and spectroscopic characterization of mutants individually disrupted in five nuo genes, equivalent to mitochondrial genes nd1, nd2, nd5, nd6 and nd4L. Disruption of any of these genes in R. capsulatus leads to the suppression of NADH dehydrogenase activity at the level of the bacterial membranes and to the disappearance of complex I‐associated iron–sulphur clusters. Individual NUO subunits can still be immunodetected in the membranes of these mutants, but they do not form a functional subcomplex. In contrast to these observations, disruption of two ORFs (orf6 and orf7 ), also present in the distal part of the nuo operon, does not suppress NADH dehydrogenase activity or complex I‐associated EPR signals, thus demonstrating that these ORFs are not essential for the biosynthesis of complex I.