Carola Hunte

Cardiolipin Stabilizes Respiratory Chain Supercomplexes

Cardiolipin stabilized supercomplexes of Saccharomyces cerevisiae respiratory chain complexes III and IV (ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase, respectively), but was not essential for their formation in the inner mitochondrial membrane because they were found also in a cardiolipin-deficient strain. Reconstitution with cardiolipin largely restored wild-type stability. The putative interface of complexes III and IV comprises transmembrane helices of cytochromes b and c1 and tightly bound cardiolipin. Subunits Rip1p, Qcr6p, Qcr9p, Qcr10p, Cox8p, Cox12p, and Cox13p and cytochrome c were not essential for the assembly of supercomplexes; and in the absence of Qcr6p, the formation of supercomplexes was even promoted. An additional marked effect of cardiolipin concerns cytochrome c oxidase. We show that a cardiolipin-deficient strain harbored almost inactive resting cytochrome c oxidase in the membrane. Transition to the fully active pulsed state occurred on a minute time scale.

Structure of a Na+/H+ antiporter and insights into mechanism of action and regulation by pH

The control by Na+/H+ antiporters of sodium/proton concentration and cell volume is crucial for the viability of all cells. Adaptation to high salinity and/or extreme pH in plants and bacteria or in human heart muscles requires the action of Na+/H+ antiporters. Their activity is tightly controlled by pH. Here we present the crystal structure of pH-downregulated NhaA, the main antiporter of Escherichia coli and many enterobacteria. A negatively charged ion funnel opens to the cytoplasm and ends in the middle of the membrane at the putative ion-binding site. There, a unique assembly of two pairs of short helices connected by crossed, extended chains creates a balanced electrostatic environment. We propose that the binding of charged substrates causes an electric imbalance, inducing movements, that permit a rapid alternating-access mechanism. This ion-exchange machinery is regulated by a conformational change elicited by a pH signal perceived at the entry to the cytoplasmic funnel.

Specific roles of protein–phospholipid interactions in the yeast cytochrome bc1 complex structure

Biochemical data have shown that specific, tightly bound phospholipids are essential for activity of the cytochrome bc1 complex (QCR), an integral membrane protein of the respiratory chain. However, the structure and function of such phospholipids are not yet known. Here we describe five phospholipid molecules and one detergent molecule in the X‐ray structure of yeast QCR at 2.3 Å resolution. Their individual binding sites suggest specific roles in facilitating structural and functional integrity of the enzyme. Interestingly, a phosphatidylinositol molecule is bound in an unusual interhelical position near the flexible linker region of the Rieske iron–sulfur protein. Two possible proton uptake pathways at the ubiquinone reduction site have been identified: the E/R and the CL/K pathway. Remarkably, cardiolipin is positioned at the entrance to the latter. We propose that cardiolipin ensures structural integrity of the proton‐conducting protein environment and takes part directly in proton uptake. Site‐directed mutagenesis of ligating residues confirmed the importance of the phosphatidylinositol‐ and cardiolipin‐binding sites.

Functional Modules and Structural Basis of Conformational Coupling in Mitochondrial Complex I

Complex I Under Scrutiny Mitochondrial complex I is a large macromolecular membrane complex that couples electron transfer to proton pumping across the mitochondrial membrane and helps to drive adenosine 5′-triphosphate synthesis. Hunte et al. (p. 448, published online 1 July) now describe the structure of complex 1 from the aerobic yeast, Yarrowia lipolytica. The sites involved in redox chemistry are distant from those that pump protons, and the structure suggests that a 60-angstrom-long helix is involved in transducing energy to the proton-pumping elements. A long helix transduces conformational energy to the proton-pumping elements in complex I. Proton-pumping respiratory complex I is one of the largest and most complicated membrane protein complexes. Its function is critical for efficient energy supply in aerobic cells, and malfunctions are implicated in many neurodegenerative disorders. Here, we report an x-ray crystallographic analysis of mitochondrial complex I. The positions of all iron-sulfur clusters relative to the membrane arm were determined in the complete enzyme complex. The ubiquinone reduction site resides close to 30 angstroms above the membrane domain. The arrangement of functional modules suggests conformational coupling of redox chemistry with proton pumping and essentially excludes direct mechanisms. We suggest that a ~60-angstrom-long helical transmission element is critical for transducing conformational energy to proton-pumping elements in the distal module of the membrane arm.

Crystal structure of the yeast cytochrome bc1 complex with its bound substrate cytochrome c

Small diffusible redox proteins facilitate electron transfer in respiration and photosynthesis by alternately binding to integral membrane proteins. Specific and transient complexes need to be formed between the redox partners to ensure fast turnover. In respiration, the mobile electron carrier cytochrome c shuttles electrons from the cytochrome bc1 complex to cytochrome c oxidase. Despite extensive studies of this fundamental step of energy metabolism, the structures of the respective electron transfer complexes were not known. Here we present the crystal structure of the complex between cytochrome c and the cytochrome bc1 complex from Saccharomyces cerevisiae. The complex was crystallized with the help of an antibody fragment, and its structure was determined at 2.97-Å resolution. Cytochrome c is bound to subunit cytochrome c1 of the enzyme. The tight and specific interactions critical for electron transfer are mediated mainly by nonpolar forces. The close spatial arrangement of the c-type hemes unexpectedly suggests a direct and rapid heme-to-heme electron transfer at a calculated rate of up to 8.3 × 106 s−1. Remarkably, cytochrome c binds to only one recognition site of the homodimeric multisubunit complex. Interestingly, the occupancy of quinone in the Qi site is higher in the monomer with bound cytochrome c, suggesting a coordinated binding and reduction of both electron-accepting substrates. Obviously, cytochrome c reduction by the cytochrome bc1 complex can be regulated in response to respiratory conditions.

Mechanistic insight from the crystal structure of mitochondrial complex I

Energy conversion in complex 1 ATP, the energy source of the cell, is synthesized by a protein residing in the mitochondrial inner membrane. The synthesis is driven by a proton gradient generated by redox reactions that transfer electrons between a series of enzymes in the membrane. The largest complex in this electron transfer chain is the 1-MD complex 1. It couples electron transfer from NADH to ubiquinone to the translocation of four protons. Zickermann et al. report the crystal structure of a complex comprising the 14 central subunits and the largest accessory subunit of mitochondrial complex 1 from a yeast-genetic model at 3.6 Å resolution. The structure identifies four potential proton translocation pathways and gives insight into how energy from the redox reactions is transmitted to drive proton pumping. Science, this issue p. 44 The x-ray structure of a protein complex shows how this enzyme pumps protons across the mitochondrial membrane. Proton-pumping complex I of the mitochondrial respiratory chain is among the largest and most complicated membrane protein complexes. The enzyme contributes substantially to oxidative energy conversion in eukaryotic cells. Its malfunctions are implicated in many hereditary and degenerative disorders. We report the x-ray structure of mitochondrial complex I at a resolution of 3.6 to 3.9 angstroms, describing in detail the central subunits that execute the bioenergetic function. A continuous axis of basic and acidic residues running centrally through the membrane arm connects the ubiquinone reduction site in the hydrophilic arm to four putative proton-pumping units. The binding position for a substrate analogous inhibitor and blockage of the predicted ubiquinone binding site provide a model for the “deactive” form of the enzyme. The proposed transition into the active form is based on a concerted structural rearrangement at the ubiquinone reduction site, providing support for a two-state stabilization-change mechanism of proton pumping.

Crystallisation of membrane proteins mediated by antibody fragments.

X-ray structures of three different membrane proteins in complex with antibody fragments have been published. The binding of Fv or Fab fragments enlarges the hydrophilic part of integral membrane proteins, thereby providing additional surface for crystal contacts and space for the detergent micelle. In all reported cases, antibody binding was either essential for the crystallisation of the membrane protein or it substantially improved the diffraction quality of the crystals. Antibody-fragment-mediated crystallisation appears to be a valuable tool in particular for membrane proteins with very small hydrophilic or flexible domains.

Protonmotive pathways and mechanisms in the cytochrome bc1 complex

The cytochrome bc 1 complex catalyzes electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which electron transfer is linked to proton translocation across the inner mitochondrial membrane. In the Q cycle mechanism proton translocation is the net result of topographically segregated reduction of quinone and reoxidation of quinol on opposite sides of the membrane, with protons being carried across the membrane as hydrogens on the quinol. The linkage of proton chemistry to electron transfer during quinol oxidation and quinone reduction requires pathways for moving protons to and from the aqueous phase and the hydrophobic environment in which the quinol and quinone redox reactions occur. Crystal structures of the mitochondrial cytochrome bc 1 complexes in various conformations allow insight into possible proton conduction pathways. In this review we discuss pathways for proton conduction linked to ubiquinone redox reactions with particular reference to recently determined structures of the yeast bc 1 complex.

Structure of complex III with bound cytochrome c in reduced state and definition of a minimal core interface for electron transfer.

In cellular respiration, cytochrome c transfers electrons from cytochrome bc1 complex (complex III) to cytochrome c oxidase by transiently binding to the membrane proteins. Here, we report the structure of isoform-1 cytochrome c bound to cytochrome bc1 complex at 1.9Å resolution in reduced state. The dimer structure is asymmetric. Monovalent cytochrome c binding is correlated with conformational changes of the Rieske head domain and subunit QCR6p and with a higher number of interfacial water molecules bound to cytochrome c1. Pronounced hydration and a “mobility mismatch” at the interface with disordered charged residues on the cytochrome c side are favorable for transient binding. Within the hydrophobic interface, a minimal core was identified by comparison with the novel structure of the complex with bound isoform-2 cytochrome c. Four core interactions encircle the heme cofactors surrounded by variable interactions. The core interface may be a feature to gain specificity for formation of the reactive complex.

Lipids and membrane protein structures

Membrane proteins do not work alone. The interaction of proteins with membrane lipids can be highly specific and is often important for full functional and structural integrity of the protein. Providing the appropriate lipid environment is of great importance for the purification and crystallisation of membrane proteins. The lipid content can be modulated by adjusting purification protocols or by adding back native or non-native lipids. Lipids can facilitate crystallisation by stabilising the protein and by providing lattice contacts. Of special interest is the crystallisation in lipidic cubic phase and with bicelles, as they appear to provide a membrane-like environment. These strategies have been instrumental for recent successful structure determinations of a human G-protein-coupled receptor, the beta(2)-adrenergic receptor. Lipid supplementation can also help to obtain membrane protein structures in a native conformation, as shown for voltage-gated potassium channels. Membrane protein structures, especially those derived from lipid-enriched preparations, contain bound lipid molecules. Specific protein-lipid interactions not only require careful evaluation and interpretation, but also permit a directed approach to elucidate the structural and/or functional role of these interactions.