J. Biol. Chem. 260, 2458 -2467). However, these assemblies did not comprise complex I (NADH:ubiquinone oxidoreductase). Using the mild detergent digitonin for solubilization of Paracoccus denitrificans membranes we could isolate NADH oxidase, assembled from complexes I, III, and IV in a 1:4:4 stoichiometry. This is the first chromatographic isolation of a complete "respirasome." Inactivation of the gene for tightly bound cytochrome c 552 did not prevent formation of this supercomplex, indicating that this electron carrier protein is not essential for structurally linking complexes III and IV. Complex I activity was also found in the membranes of mutant strains lacking complexes III or IV. However, no assembled complex I but only dissociated subunits were observed following the same protocols used for electrophoretic separation or chromatographic isolation of the supercomplex from the wild-type strain. This indicates that the P. denitrificans complex I is stabilized by assembly into the NADH oxidase supercomplex. In addition to substrate channeling, structural stabilization of a membrane protein complex thus appears as one of the major functions of respiratory chain supercomplexes.

We previously proposed that the dimeric cytochrome bc 1 complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc 1 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 bc 1 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 c 1 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 wildtype 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 cytochrome bc 1 complex is a multisubunit enzyme that generates a transmembrane protonmotive force by transferring electrons from ubiquinol to cytochrome c. Energy conservation in this enzyme complex is ensured by the oxidation of ubiquinol and the reduction of ubiquinone at active sites located on opposite sides of the membrane, as described by the protonmotive Q-cycle (1). High resolution structures have shown that the bc 1 complex is a dimer composed of two copies of cytochrome b, the Rieske iron-sulfur protein, and cytochrome c 1 , which are the only polypeptides present in some bacterial enzymes (2), in addition to six to eight additional subunits present exclusively in each monomer of the eukaryotic complex (3-5).The functional relevance of this dimeric arrangement has been supported by an extensive body of kinetic evidence (for review, see Ref. 6), which includes the key observations that only one ubiquinol oxidation site, or center P, is active when the two ubiquinone reduction, or center N, sites are occupied by inhibitors (7), that electrons rapidly equilibrate between the cytochrome b subunits (8, 9), and that there is conformational communication between center P and center N sites (10). We have proposed that this half-of-the sites activity at center P also exists under normal steady-state conditions in the absence of inhibitors and that this mechanism minimizes the leakage of electrons to oxygen under conditions that would favor the accumulation of electrons in the cytochrome b hemes (8, 11). Evidence consistent with this...

The cytochrome bc(1) complex from Paracoccus denitrificans and soluble fragments of its cytochrome c(1) and Rieske ISP subunits are characterized by a combined approach of protein electrochemistry and FTIR difference spectroscopy. The FTIR difference spectra provide information about alterations in the protein upon redox reactions: signals from the polypeptide backbone, from the cofactors, and from amino acid side chains. We describe typical modes for conformational changes in the polypeptide and contributions of different secondary structure elements. Signals attributed to the different cofactors can be presented on the basis of selected potential steps. Modes associated with bound quinone are identified by comparison with spectra of quinone in solution at 1656, 1642, and 1610 cm(-1) and between 1494 and 1388 cm(-1), as well as at 1288 and 1262 cm(-1). Signals originating from the quinone bound at the Q(o) site can be distinguished. On the basis of the infrared data, the total quinone concentration is determined to be 2.6-3.3 quinones per monomer, depending on preparation conditions. The balance of evidence supports the double-occupancy model. Interestingly, the amplitude of the band at 1746 cm(-1) increases with quinone content, reflecting a protonation reaction of acidic groups. In this context, the involvement of glutamates and/or aspartates in the vicinity of the Q(o) site is discussed on the basis of recently determined crystal structures.

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 K d 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.

Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic ␤-cells, and small molecule activators of GK (GKAs) are under evaluation for the treatment of type 2 diabetes. In liver, GK activity is controlled by the GK regulatory protein (GKRP), which forms an inhibitory complex with the enzyme. Here, we performed isothermal titration calorimetry and surface plasmon resonance experiments to characterize GK-GKRP binding and to study the influence that physiological and pharmacological effectors of GK have on the protein-protein interaction. In the presence of fructose-6-phosphate, GK-GKRP complex formation displayed a strong entropic driving force opposed by a large positive enthalpy; a negative change in heat capacity was observed (K d ‫؍‬ 45 nM, ⌬H ‫؍‬ 15.6 kcal/mol, T⌬S ‫؍‬ 25.7 kcal/mol, ⌬C p ‫؍‬ ؊354 cal mol ؊1 K ؊1 ). With k off ‫؍‬ 1.3 ؋ 10 ؊2 s ؊1 , the complex dissociated quickly. The thermodynamic profile suggested a largely hydrophobic interaction. In addition, effects of pH and buffer demonstrated the coupled uptake of one proton and indicated an ionic contribution to binding. Glucose decreased the binding affinity between GK and GKRP. This decrease was potentiated by an ATP analogue. Prototypical GKAs of the amino-heteroaryl-amide type bound to GK in a glucose-dependent manner and impaired the association of GK with GKRP. This mechanism might contribute to the antidiabetic effects of GKAs. Glucokinase (GK)2 is the predominant glucose-phosphorylating enzyme in liver and pancreatic ␤-cells and plays a central role in blood glucose homeostasis (1, 2). Enhancing GK activity by small molecule GK activators (GKAs) is currently under evaluation as an approach for the treatment of diabetes (3). Hepatic GK activity is controlled by an endogenous inhibitor, a 68-kDa GK regulatory protein (GKRP) (4). During starvation, the enzyme is bound to GKRP, leading to its inactivation and sequestration in the nucleus. After refeeding, the GK-GKRP complex dissociates and GK translocates into the cytoplasm (5, 6). In a rodent model of type 2 diabetes, this translocation is impaired, which could contribute to the defective blood glucose homeostasis (7). Further evidence for the metabolic impact of GKRP comes from recent human genome-wide association studies that show a strong linkage between a GKRP gene polymorphism and serum triglyceride levels (8, 9). The formation of the GK-GKRP complex is favored by fructose-6-phosphate (F6P) and inhibited by fructose-1-phosphate (F1P) (10). Both sugar phosphates bind to the same site on the regulatory protein (11). An increase of the intracellular F1P concentration leading to the release of GK from its inhibitory complex with GKRP is most likely the reason for the stimulation of hepatic glucose metabolism by catalytic amounts of fructose or sorbitol (6, 12, 13). Site-directed mutagenesis experiments indicate that the binding interface for GKRP lies close to the binding site of allosteric GKAs of the amino-heteroaryl-amide type (14). However, it is controversial if thes...

Mitochondrial respiratory complex III ⁄ cytochrome bc 1 is among the best-characterized membrane proteins, with structures elucidated from several species [1][2][3][4]. These structures revealed the organization of three catalytic subunits (SU) in the homodimeric complex; these are cytochrome b, cytochrome c 1 , and the Rieske ironsulfur protein (ISP). Cytochrome b forms eight transmembrane (TM) helices that bind two hemes and is largely contained within the membrane bilayer. Both cytochrome c 1 and the ISP are single-spanning TM proteins with globular hydrophilic domains located in the periplasmic space; these ecto-domains carry the heme and [2Fe)2S] cofactors, respectively. As the first cytochrome bc 1 structures were characterized, two features of the ISP came as a major surprise: (a) the ISP intertwines between the monomeric halves of the enzyme, such that the N-terminal TM helix of a given ISP is anchored within one monomer, whereas the The Rieske [2Fe)2S] protein (ISP) is an essential subunit of cytochrome bc 1 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 bc 1 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.Abbreviations EPR, electron paramagnetic resonance spectroscopy; ISF, Rieske iron-sulfur protein soluble fragment; ISP, Rieske iron-sulfur protein; SU, subunit; Tat, twin-arginine translocation; TM, transmembrane.

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Glycogen phosphorylase (GP) is a validated target for the treatment of type 2 diabetes. Here we describe highly potent GP inhibitors, AVE5688, AVE2865, and AVE9423. The first two compounds are optimized members of the acyl urea series. The latter represents a novel quinolone class of GP inhibitors, which is introduced in this study. In the enzyme assay, both inhibitor types compete with the physiological activator AMP and act synergistically with glucose. Isothermal titration calorimetry (ITC) shows that the compounds strongly bind to nonphosphorylated, inactive GP (GPb). Binding to phosphorylated, active GP (GPa) is substantially weaker, and the thermodynamic profile reflects a coupled transition to the inactive (tense) conformation. Crystal structures confirm that the three inhibitors bind to the AMP site of tense state GP. These data provide the first direct evidence that acyl urea and quinolone compounds are allosteric inhibitors that selectively bind to and stabilize the inactive conformation of the enzyme. Furthermore, ITC reveals markedly different thermodynamic contributions to inhibitor potency that can be related to the binding modes observed in the cocrystal structures. For AVE5688, which occupies only the lower part of the bifurcated AMP site, binding to GPb (Kd = 170 nM) is exclusively enthalpic (Delta H = -9.0 kcal/mol, TDelta S = 0.3 kcal/mol). The inhibitors AVE2865 (Kd = 9 nM, Delta H = -6.8 kcal/mol, TDelta S = 4.2 kcal/mol) and AVE9423 (Kd = 24 nM, Delta H = -5.9 kcal/mol, TDelta S = 4.6 kcal/mol) fully exploit the volume of the binding pocket. Their pronounced binding entropy can be attributed to the extensive displacement of solvent molecules as well as to ionic interactions with the phosphate recognition site.