Neal C. Robinson

Functional binding of cardiolipin to cytochrome c oxidase

Bovine cytochromec oxidase usually contains 3–4 mol of tightly bound cardiolipin per cytochromeaa3 complex. At least two of these cardiolipins are required for full electron transport activity. Without the tightly bound cardiolipin, cytochromec oxidase has only 40–50% of its original activity when assayed in detergents that support activity, e.g., dodecyl maltoside. By measuring the restoration of electron transport activity, functional binding constants for cardiolipin and a number of cardiolipin analogues have been evaluated (Kd,app=1 µM for cardiolipin). These binding constants agree reasonably well with direct measurement of the binding using [14C]-acetyl-cardiolipin (Kd<0.1 µM) when the enzyme is solubilized with Triton X-100. These data are discussed in relationship to the wealth of data that is known about the association of cardiolipin with cytochromec oxidase and the other mitochrondrial electron transport complexes and transporters.

Tryptophan 334 Oxidation in Bovine Cytochrome c Oxidase Subunit I Involves Free Radical Migration

A single tryptophan (W334(I)) within the mitochondrial‐encoded core subunits of cytochrome c oxidase (CcO) is selectively oxidized when hydrogen peroxide reacts with the binuclear center. W334(I) is converted to hydroxytryptophan as identified by reversed‐phase HPLC‐electrospray ionization tandem mass spectrometry analysis of peptides derived from the three SDS–PAGE purified subunits. Total sequence coverage of subunits I, II and III was limited to 84%, 66% and 54%, respectively. W334(I) is located on the surface of CcO at the membrane interface. Two other surface tryptophans within nuclear‐encoded subunits, W48(IV) and W19(VIIc), are also oxidized when hydrogen peroxide reacts with the binuclear center (Musatov et al. (2004) Biochemistry 43, 1003–1009). Two aromatic‐rich networks of amino acids were identified that link the binuclear center to the three oxidized tryptophans. We propose the following mechanism to explain these results. Electron transfer through the aromatic networks moves the free radicals generated at the binuclear center to the surface‐exposed tryptophans, where they produce hydroxytryptophan.

Dansylation of bacteriorhodopsin near the retinal attachment site.

Abstract The purple membrane of Halobacterium halobium was reacted with 5-dimethylaminonaphthalene-l-sulfonyl chloride (dansyl chloride) at pH 8.0. Chromophoric and functional properties of the product appear unaltered. Approximately 2 moles of dansyl group were incorporated per mole of bacteriorhodopsin, part bound to bacteriorhodopsin and part bound to lipids. Purification and fragmentation of the protein showed most of the dansyl modification in a fragment containing residues 33 to 56. Amino acid analysis indicates that the major dansylated site is lysine 40. We conclude that, contrary to published models, 1) bacteriorhodopsin folds in a way that exposes lysine 40 at the membrane surface, and 2) this side chain is not involved in the proton pump mechanism.

Subunit analysis of bovine cytochrome c oxidase by reverse phase high performance liquid chromatography

Bovine cytochrome c oxidase subunits were separated by reverse phase high performance liquid chromatography using a C4 column eluted with water and an acetonitrile gradient, both containing 0.1% trifluoroacetic acid. Subunits I and III precipitated in this solvent and could not be analyzed; the remaining eleven subunits were dissociated, denatured, soluble and could be resolved by elution from the column. The protein subunit eluting in each chromatographic peak was identified by a combination of polyacrylamide gel electrophoresis in sodium dodecyl sulfate, NH2-terminal amino acid sequencing, and amino acid analysis. Each subunit produced a single elution peak with the exception of subunit VIc (nomenclature of Kadenbach et al., 1983, Anal. Biochem. 129, 517-521), which eluted from the column as two well-resolved peaks. Sequence analysis showed that the two subunit VIc elution peaks resulted from partial chemical blockage of the alpha-amino serine residue of subunit VIc. The C4 reverse phase HPLC was used to document specific subunit removal from bovine cytochrome c oxidase either by tryptic digestion or by dodecyl maltoside extraction. The described HPLC method for separating cytochrome c oxidase subunits should be applicable for the analysis of other multisubunit proteins, especially other multisubunit membrane protein complexes.

Sequential Dissociation of Subunits from Bovine Heart Cytochrome c Oxidase by Urea

The quaternary stability of purified, detergent-solubilized, cytochrome c oxidase (CcO) was probed using two chemical denaturants, urea and guanidinium chloride (GdmCl). Each chaotrope induces dissociation of five subunits in a concentration-dependent manner. These five subunits are not scattered over the surface of CcO but are clustered together in close contact at the dimer interface. Increasing the concentration of urea selectively dissociates subunits from CcO in the following order: VIa and VIb, followed by III and VIIa, and finally Vb. After incubation in urea for 10 min at room temperature, the sigmoidal dissociation transitions were centered at 3.7, 4.6, and 7.0 M urea, respectively. The secondary structure of CcO was only minimally perturbed, indicating that urea causes disruption of subunit interactions without urea-induced conformational changes. Incubation of CcO in urea for 120 min produced similar results but shifted the sigmoidal dissociation curves to lower urea concentrations. Incubation of CcO with increasing concentrations of GdmCl produces an analogous effect; however, the GdmCl-induced dissociation of subunits occurs at lower concentrations and with a narrower concentration range. Thermodynamic parameters for each subunit dissociation were evaluated from the sigmoidal dissociation data by assuming a single transition from bound to dissociated subunit. The free energy change accompanying urea-induced dissociation of each subunit ranged from 18.0 to 29.7 kJ/mol, which corresponds to 0.32-0.59 kJ/mol per 100 A(2) of newly exposed solvent-accessible surface area. These values are 30-50-fold smaller than previously reported for the unfolding of soluble or membrane proteins.

Subunit analysis of bovine heart complex I by reversed-phase high-performance liquid chromatography, electrospray ionization–tandem mass spectrometry, and matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry

An effective method was developed for isolation and analysis of bovine heart complex I subunits. The method uses C18 reversed-phase high-performance liquid chromatography (HPLC) and a water/acetonitrile gradient containing 0.1% trifluoroacetic acid. Employing this system, 36 of the 45 complex I subunits elute in 28 distinct chromatographic peaks. The 9 subunits that do not elute are B14.7, MLRQ, and the 7 mitochondrial-encoded subunits. The method, with ultraviolet (UV) detection, is suitable for either analytical (<50 microg protein) or preparative (>250 microg protein) applications. Subunits eluting in each chromatographic peak were initially determined by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) with subsequent positive identification by reversed-phase HPLC-electrospray ionization (ESI)/tandem mass spectrometry (MS/MS) analysis of tryptic digests. In the latter case, subunits were identified with a 99% probability using Mascot for database searching and Scaffold for assessment of protein identification probabilities. The reversed-phase HPLC subunit analysis method represents a major improvement over previous separation methods with respect to resolution, simplicity, and ease of application.

DETERGENT-SOLUBILIZED ESCHERICHIA COLI CYTOCHROME BO3 UBIQUINOL OXIDASE : A MONOMERIC, NOT A DIMERIC COMPLEX

The protein molecular weight, M r, and hydrodynamic radius, R s, of Triton X‐100‐solubilized Escherichia coli cytochrome bo 3 were evaluated by computer fitting of sedimentation velocity data with finite element solutions to the Lamm equation. Detergent‐solubilized cytochrome bo 3 sediments as a homogeneous species with an s 20,w of 6.75 s and a D 20,w of 3.71×10−7 cm2/s, corresponding to a R s of 5.8 nm and a M r of 144 000±3500. The protein molecular weight agrees very well with the value of 143 929 calculated from the four known subunit sequences and the value of 143 025 measured by MALDI mass spectrometry for the histidine‐tagged enzyme. We conclude that detergent‐solubilized E. coli ubiquinol oxidase is a monomeric complex of the four known subunits.

Diphosphatidylglycerol Reactivation of Lipid-Depleted Beef Heart Cytochrome c Oxidase: Effect of Subunit III Removal

Beef heart cytochrome c oxidase can be fully delipidated by Triton X-100 using an alkaline pH, high ionic strength incubation followed by glycerol gradient centrifugation at pH 8 in 1% Triton X-100. Unfortunately, this procedure removes not only the 2-3 diphosphatidylglycerol (DPG) molecules that are tightly bound to each heme aa3 complex, but also removes subunit III. A one-to-one correlation exists between the percent of subunit III removed and the percent of tightly bound DPG extracted, suggesting a possible tight binding of these two molecules. However, based upon regeneration of most of the electron transport activity of the subunit III deficient complex by exogenous DPG, it appears that the functional DPG binding site must be located on a subunit other than subunit III.

Membrane proteins: a summary of known structural information.

Publisher Summary This chapter focuses on membrane proteins. The knowledge concerning the structure of intrinsic membrane proteins has greatly advanced during the past decade due to the purification and characterization of hundreds of these proteins. However, much of the information about the three-dimensional structure, size, shape, molecular weight, amino acid sequence, and amino acid composition of these proteins is scattered throughout the literature making comparisons difficult. To facilitate the study of this class of proteins, this chapter presents a list of the intrinsic membrane proteins that are purified and reviews articles or literature reports containing structural information. These proteins are divided into two categories based on the extent to which they have been characterized: (1) the proteins that have been well characterized, for example, those proteins for which a reasonably accurate structural model has been determined; and (2) the proteins, for example, that have only been purified or for which limited structural information is available.

Removal of bound Triton X-100 from purified bovine heart cytochrome bc1

Cytochrome bc(1) isolated from Triton X-100-solubilized mitochondrial membranes contains up to 120 nmol of Triton X-100 bound per nanomole of the enzyme. Purified cytochrome bc(1) is fully active; however, protein-bound Triton X-100 significantly interferes with structural studies of the enzyme. Removal of Triton X-100 bound to bovine cytochrome bc(1) was accomplished by incubation with Bio-Beads SM-2 in the presence of sodium cholate. Sodium cholate is critical because it does not interfere with the adsorption of protein on the hydrophobic surface of the beads. The resulting Triton X-100-free cytochrome bc(1) retained nearly full activity, absorption spectra, subunit, and phospholipid composition.