Ruth Hielscher

Role of phospholipids in respiratory cytochrome bc1 complex catalysis and supercomplex formation

Specific protein-lipid interactions have been identified in X-ray structures of membrane proteins. The role of specifically bound lipid molecules in protein function remains elusive. In the current study, we investigated how phospholipids influence catalytic, spectral and electrochemical properties of the yeast respiratory cytochrome bc(1) complex and how disruption of a specific cardiolipin binding site in cytochrome c(1) alters respiratory supercomplex formation in mitochondrial membranes. Purified yeast cytochrome bc(1) complex was treated with phospholipase A(2). The lipid-depleted enzyme was stable but nearly catalytically inactive. The absorption maxima of the reduced b-hemes were blue-shifted. The midpoint potentials of the b-hemes of the delipidated complex were shifted from -52 to -82 mV (heme b(L)) and from +113 to -2 mV (heme b(H)). These alterations could be reversed by reconstitution of the delipidated enzyme with a mixture of asolectin and cardiolipin, whereas addition of the single components could not reverse the alterations. We further analyzed the role of a specific cardiolipin binding site (CL(i)) in supercomplex formation by site-directed mutagenesis and BN-PAGE. The results suggested that cardiolipin stabilizes respiratory supercomplex formation by neutralizing the charges of lysine residues in the vicinity of the presumed interaction domain between cytochrome bc(1) complex and cytochrome c oxidase. Overall, the study supports the idea, that enzyme-bound phospholipids can play an important role in the regulation of protein function and protein-protein interaction.

Heterologous Production, Isolation, Characterization and Crystallization of a Soluble Fragment of the NADH:Ubiquinone Oxidoreductase (Complex I) from Aquifex aeolicus†

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) is the first enzyme complex of the respiratory chains in many bacteria and most eukaryotes. It is the least understood of all, due to its enormous size and unique energy conversion mechanism. The bacterial complex is in general made up of 14 different subunits named NuoA-N. Subunits NuoE, -F, and -G comprise the electron input part of the complex. We have cloned these genes from the hyperthermophilic bacterium Aquifex aeolicus and expressed them heterologously in Escherichia coli. A soluble subcomplex made up of NuoE and NuoF and containing the NADH binding site, the primary electron acceptor flavin mononucleotide (FMN), the binuclear iron-sulfur cluster N1a, and the tetranuclear iron-sulfur cluster N3 was isolated by chromatographic methods. The proteins were identified by N-terminal sequencing and mass spectrometry; the cofactors were characterized by UV/vis and EPR spectroscopy. Subunit NuoG was not produced in this strain. The preparation was thermostable and exhibited maximum NADH/ferricyanide oxidoreductase activity at 85 degrees C. Analytical size-exclusion chromatography and dynamic light scattering revealed the homogeneity of the preparation. First attempts to crystallize the preparation led to crystals diffracting more than 2 A.

Nucleotide-induced conformational changes in the Escherichia coli NADH:ubiquinone oxidoreductase (complex I).

The energy-converting NADH:ubiquinone oxidoreductase, also known as respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy revealed the two-part structure of the complex consisting of a peripheral and a membrane arm. The peripheral arm contains all known cofactors and the NADH-binding site, whereas the membrane arm has to be involved in proton translocation. Owing to this, a conformation-linked mechanism for redox-driven proton translocation is discussed. By means of electron microscopy, we show that both arms of the Escherichia coli complex I are widened after the addition of NADH but not of NADPH. NADH-induced conformational changes were also detected in solution: ATR-FTIR (attenuated total reflection Fourier-transform infrared) of the soluble NADH dehydrogenase fragment of the complex indicates protein re-arrangements induced by the addition of NADH. EPR spectroscopy of surface mutants of the complex containing a covalently bound spin label at distinct positions demonstrates NADH-dependent conformational changes in both arms of the complex.

Far‐ and Mid‐Infrared Spectroscopic Analysis of the Substrate‐Induced Structural Dynamics of Respiratory Complex I

The catalytic activity of the respiratory NADH:ubiquinone oxidoreductase (complex I) is based on conformational reorganizations. Herein we probe the effect of substrates on the conformational flexibility of complex I by means of (1)H/(2)H exchange kinetics at the level of the amide proton in the mid-infrared spectral range (1700-1500 cm(-1)). Slow, medium, and fast exchanging domains are distinguished that reveal different accessibilities to the solvent. Whereas amide hydrogens undergo rapid exchange with the solvent in an open structure, hydrogens experience much slower exchange when they are involved in H-bonded structures or when they are sterically inaccessible for the solvent. The results indicate a structure that is more open in the presence of both NADH and quinon. Complementary information on the overall internal hydrogen bonding of the protein was probed in the far infrared (300-30 cm(-1)), a spectral range that includes a continuum mode of the hydrogen bonding signature.

Monitoring the redox and protonation dependent contributions of cardiolipin in electrochemically induced FTIR difference spectra of the cytochrome bc1 complex from yeast

Biochemical studies have shown that cardiolipin is essential for the integrity and activity of the cytochrome bc(1) complex and many other membrane proteins. Recently the direct involvement of a bound cardiolipin molecule (CL) for proton uptake at center N, the site of quinone reduction, was suggested on the basis of a crystallographic study. In the study presented here, we probe the low frequency infrared spectroscopy region as a technique suitable to detect the involvement of the lipids in redox induced reactions of the protein. First the individual infrared spectroscopic features of lipids, typically present in the yeast membrane, have been monitored for different pH values in micelles and vesicles. The pK(a) values for cardiolipin molecule have been observed at 4.7+/-0.3 and 7.9+/-1.3, respectively. Lipid contributions in the electrochemically induced FTIR spectra of the bc(1) complex from yeast have been identified by comparing the spectra of the as isolated form, with samples where the lipids were digested by lipase-A(2). Overall, a noteworthy perturbation in the spectral region typical for the protein backbone can be reported. Interestingly, signals at 1159, 1113, 1039 and 980 cm(-1) have shifted, indicating the perturbation of the protonation state of cardiolipin coupled to the reduction of the hemes. Additional shifts are found and are proposed to reflect lipids reorganizing due to a change in their direct environment upon the redox reaction of the hemes. In addition a small shift in the alpha band from 559 to 556 nm can be seen after lipid depletion, reflecting the interaction with heme b(H) and heme c. Thus, our work highlights the role of lipids in enzyme reactivity and structure.

Multiple step assembly of the transmembrane cytochrome b6.

We have analyzed the role of individual heme-ligating histidine residues for assembly of holo-cytochrome b(6), and we show that the two hemes b(L) and b(H) bind in two subsequent steps to the apo-protein. Binding of the low-potential heme b(L) is a prerequisite for binding the high-potential heme b(H). After substitution of His86, which serves as an axial ligand for heme b(L), the apo-protein did not bind heme, while substitution of the heme b(L)-ligating residue His187 still allowed binding of both hemes. Similarly, after replacement of His202, one axial ligand to heme b(H), binding of only heme b(L) was observed, whereas replacement of His100, the other heme b(H) ligand, resulted in binding of both hemes. These data indicate sequential heme binding during formation of the holo-cytochrome, and the two histidine residues, which serve as axial ligands to the same heme molecule (heme b(L) or heme b(H)), have different importance during heme binding and cytochrome assembly. Furthermore, determination of the heme midpoint potentials of the various cytochrome b(6) variants indicates a cooperative adjustment of the heme midpoint potentials in cytochrome b(6).

Characterization of two cytochrome b6 proteins from the cyanobacterium Gloeobacter violaceus PCC 7421.

In the genome of the untypical cyanobacterium Gloeobacter violaceus PCC 7421 two potential cytochrome b6 proteins PetB1 and PetB2 are encoded. Such a situation has not been observed in cyanobacteria, algae and higher plants before, and both proteins are not characterized at all yet. Here, we show that both apo-proteins bind heme with high affinity and the spectroscopic characteristics of both holo-proteins are distinctive for cytochrome b6 proteins. However, while in PetB2 one histidine residue, which corresponds to H100 and serves as an axial ligand for heme bH in PetB1, is mutated, both PetB proteins bind two heme molecules with different midpoint potentials. To recreate the canonical heme bH binding cavity in PetB2 we introduced a histidine residue at the position corresponding to H100 in PetB1 and subsequently characterized the generated protein variant. The presented data indicate that two bona fide cytochrome b6 proteins are encoded in Gloeobacter violaceus. Furthermore, the two petB genes of Gloeobacter violaceus are each organized in an operon together with a petD gene. Potential causes and consequences of the petB and petD gene heterogeneity are discussed.

The Temperature‐Dependent Hydrogen‐Bonding Signature of Lipids Monitored in the Far‐Infrared Domain

Phospholipids are studied by means of Fourier transform infrared (FTIR) spectroscopy in the mid- and far-infrared spectral ranges, thereby establishing the hydrogen-bonding continuum as a function of the temperature. The well-known mid-infrared spectrum of the phospholipid layer clearly shows a temperature-dependent phase transition. In the far-infrared region (from 300 to 50 cm(-1)), an alternation of the interaction between the phospholipids and water molecules is found. The hydrogen-bonding network ensemble and bound water molecules can be monitored in this spectral region. The lipid structure is found to strongly influence the intermolecular hydrogen-bonding interplay. Thus, studies in the far-infrared region provide significant information--at the molecular level--about the intermolecular hydrogen-bonding signature of self-assembled phospholipids.

Characterization of Two Quinone Radicals in the NADH:Ubiquinone Oxidoreductase from Escherichia coli by a Combined Fluorescence Spectroscopic and Electrochemical Approach

The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. It was proposed that the electron transfer involves quinoid groups localized at the end of the electron transfer chain. To identify these groups, fluorescence excitation and emission spectra of Escherichia coli complex I and its fragments, namely, the NADH dehydrogenase fragment containing the flavin mononucleotide and six iron-sulfur (Fe-S) clusters, and the quinone reductase fragment containing three Fe-S clusters were measured. Signals sensitive to reduction by either NADH or dithionite were detected within the complex and the quinone reductase fragment and attributed to the redox transition of protonated ubiquinone radicals. A fluorescence spectroscopic electrochemical redox titration revealed midpoint potentials of -37 and- 235 mV (vs the standard hydrogen electrode) for the redox transitions of the quinone radicals in complex I at pH 6 with an absorption around 325 nm and a fluorescence emission at 460/475 nm. The role of these cofactor(s) for electron transfer is discussed.

Specific Far Infrared Spectroscopic Properties of Phospholipids

We describe the specific spectral signature of different phospholipids and sphingolipids in the far infrared. Three specific spectral domains have been found: the head group contributions (600 and 480 cm−1); the modes of the torsion motion of the hydrocarbon chains and of the skeleton vibration (460 to 180 cm−1); and the hydrogen-bonding continuum (below 300 cm−1). Marker bands for individual phospholipids are distinguished.