Dimeric Oxidase Research Articles

Interrogating the higher order structures of snake venom proteins using an integrated mass spectrometric approach

Snake venoms contain complex mixtures of proteins vital for the survival of venomous snakes. Aligned with their diverse pharmacological activities, the protein compositions of snake venoms are highly variable, and efforts to characterise the primary structures of such proteins are ongoing. Additionally, a significant knowledge gap exists in terms of the higher-order protein structures which modulate venom potency, posing a challenge for successful therapeutic applications. Here we use a multifaceted mass spectrometry approach to characterise proteins from venoms of Collett's snake Pseudechis colletti and the puff adder Bitis arietans. Following chromatographic fractionation and bottom-up proteomics analysis, native mass spectrometry identified, among other components, a non-covalent l-amino acid oxidase dimer in the P. colletti venom and a C-type lectin tetramer in the B. arietans venom. Furthermore, a covalently-linked phospholipase A2 (PLA2) dimer was identified in P. colletti venom, from which the PLA2 species were shown to adopt compact geometries using ion mobility measurements. Interestingly, we show that the dimeric PLA2 possesses greater bioactivity than the monomeric PLA2s. This work contributes to ongoing efforts cataloguing components of snake venoms, and notably, emphasises the importance of understanding higher-order venom protein interactions and the utility of a combined mass spectrometric approach for this task. SignificanceThe protein constituents of snake venoms represent a sophisticated cocktail of biologically active molecules ideally suited for further exploration in drug design and development. Despite ongoing efforts to characterise the diverse protein components of such venoms there is still much work required in this area, particularly in moving from simply describing the protein primary sequence to providing an understanding of quaternary structure. The combined proteomic and native mass spectrometry workflow utilised here gives new insights into higher order protein structures in selected snake venoms, and can underpin further investigation into the protein interactions which govern snake venom specificity and potency.

Nano-Encapsulation of Glucose Oxidase Dimer by Graphene

ABSTRACTUsing all-atom molecular dynamics simulations in water environment, it was possible to demonstrate spontaneous and tight encapsulation of glucose oxidase (GOx) dimer by graphene 7 nm x 7 nm sheets linked together by linkers of different width and forming a flower-like or cross-like shapes. The partially overlapping graphene sheets compacted the structure of GOx dimer, bringing the monomers much closer to one another. We found that the most complete wrapping of the enzyme was achieved for the cross-like graphene. Encapsulation can be a useful way to obtain a large contact surface. However, an exceptionally tight binding by the graphene can also influence the positions of amino acids in the enzyme binding site resulting in less efficient catalytic reaction. Furthermore, such extensive encapsulation could block the access of the substrate to the active site of the enzyme. Contrary, a partial encapsulation by graphene using nano-sheets caused only small distortions of GOx structure while the contact surface with graphene was high.

Molecular effects of encapsulation of glucose oxidase dimer by graphene

A box-like shape of graphene leads to different types of “sandwich” or “burrito” encapsulation of the enzyme. To preserve the critical interactions in the enzyme active site a proper balance of forces between protein and graphene is required.

Non-equivalent conformations ofd-amino acid oxidase dimer from porcine kidney between the two subunits. Molecular dynamics simulation and photoinduced electron transfer

The structural difference between two subunits of D-amino acid oxidase dimer from porcine kidney was studied by molecular dynamics simulation (MDS) and rate of photoinduced electron transfer (ET) from aromatic amino acids as tyrosines (Tyr) and tryptophanes (Trp) to the excited isoalloxazine (Iso*). The donor-acceptor distances (Rc) between isoalloxazine (Iso) and the donors were shortest in Tyr224 (0.74 nm) in Sub A at 10 °C (Sub A10), in Tyr224 (0.79 nm) in Sub B at 10 °C (Sub B10), in Tyr228 (0.85 nm) in Sub A at 30 °C (Sub A30), and in Tyr224 (0.72 nm) in Sub B at 30 °C (Sub B30). The Rcs were mostly shorter in the dimer than those in the monomer. Hydrogen bonding (H-bond) pairs between Iso and surrounding amino acids varied with the subunit and temperature. O2 of the Iso ring formed an H-bond exclusively with Thr317OG1 (side chain) in both Sub A10 and Sub A30, while it formed with Gly315N (peptide), Leu316N and Thr317N in Sub B10 and Sub B30. N3H of Iso formed an H-bond with Leu51O (peptide) in Sub A10 and Sub A30, but not in Sub B10 and Sub B30. Electron affinity of Iso* was appreciably lower in Sub A10 compared to Sub B10, while it was opposite at 30 °C. ET rate to Iso* was fastest from Tyr224 in Sub A10, while it was fastest from Tyr314 in Sub B10. The ET rate was fastest from Tyr314 in Sub A30, while it was fastest from Tyr224 in Sub B30. The greater ET rates in the dimer as compared to those in the monomer were elucidated with shorter Rc in the dimer as compared to the monomer. The static dielectric constants inside the subunits and the static dielectric constant between Iso and Tyr224 or Tyr228 were not different appreciably. A few water molecules and sometimes an amino acid were located between Iso and Tyr224, which may be the reason why the dielectric constant of the entire subunits did not differ from that between Iso and Tyr224.

Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity

Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0–400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay.

Knockdown of Human COX17 Affects Assembly and Supramolecular Organization of Cytochrome c Oxidase

Assembly of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, requires a concerted activity of a number of chaperones and factors for the insertion of subunits, accessory proteins, cofactors and prosthetic groups. It is now well accepted that the multienzyme complexes of the respiratory chain are organized in vivo as supramolecular functional structures, so-called supercomplexes. Here, we investigate the role of COX17 in the biogenesis of the respiratory chain in HeLa cells. In accordance with its predicted function as a copper chaperone and its role in formation of the binuclear copper centre of cytochrome c oxidase, COX17 siRNA knockdown affects activity and assembly of cytochrome c oxidase. While the abundance of cytochrome c oxidase dimers seems to be unaffected, blue native gel electrophoresis reveals the disappearance of COX-containing supercomplexes as an early response. We observe the accumulation of a novel ∼150 kDa complex that contains Cox1, but not Cox2. This observation may indicate that the absence of Cox17 interferes with copper delivery to Cox2, but not to Cox1. We suggest that supercomplex formation is not simply due to assembly of completely assembled complexes. An interdependent assembly scenario for the formation of supercomplexes that rather requires the coordinated synthesis and association of individual complexes, is proposed.

Lack of Formation of Aldehyde Oxidase Dimer Possibly Due to 377G>A Nucleotide Substitution

In addition to the many articles reporting on the marked differences in species and large differences in rat strains in response to aldehyde oxidase (AO), individual differences in some rat strains have also been reported. However, little has been clarified about any related molecular biological mechanisms. We previously revealed that nucleotide substitutions of 377G>A and 2604C>T in the AO gene might be responsible for individual differences in AO activity in Donryu strain rats. By using native polyacrylamide gel electrophoresis/Western blotting in this study, the lack of formation of the AO dimer protein, which is essential for catalytic activity, was shown in poor metabolizer Donryu rats, and this could be a major reason for the individual differences. Rat strain differences were also verified from the same perspectives of nucleotide substitutions and expression levels of a dimer protein. Rat strains with high AO activity showed nucleotide sequences of (377G, 2604C) and a dimer protein. In the case of those with low AO activity, the nucleotide at position 2604 was fixed at T, but varied at position 377, such as G, G/A, and A. An AO dimer was detected in the liver cytosols of rat strains with (377G, 2604T), whereas a monomer was observed in those with (377A, 2604T). These results suggest that the lack of formation of a dimer protein leading to loss of catalytic activity might be due to 377G>A nucleotide substitution. Individual and strain differences in AO activity in rats could be explained by this 377G>A substitution, at least in the rat strains used in this study.

The Cyanide-Resistant Alternative Oxidases from the Fungi Pichia stipitis and Neurospora crassa Are Monomeric and Lack Regulatory Features of the Plant Enzyme

Both plant and fungal mitochondria have cyanide-resistant alternative oxidases that use reductant from the mitochondrial ubiquinone pool to reduce oxygen to water in a reaction that conserves no energy for ATP synthesis. The dimeric plant alternative oxidase is relatively inactive when its subunits are linked by a disulfide bond. When this bond is reduced, the enzyme can then be stimulated by its activators, α-keto acids. A Cys in the N-terminal section of the protein is responsible for both of these features. We examined the alternative oxidases in mitochondria isolated from two fungi Neurospora crassa and Pichia stipitis for dimeric structure, ability to form an intermolecular disulfide, and sensitivity to α-keto acids. Neither of the two fungal alternative oxidases could be covalently linked by diamide, which induces disulfide bond formation between nearby Cys residues, nor could they be cross-linked by a Lys-specific reagent or glutaraldehyde at concentrations which cross-link the plant alternative oxidase dimer completely. Alternative oxidase activity in fungal mitochondria was not stimulated by the α-keto acids pyruvate and glyoxylate. Pyruvate did stimulate activity when succinate was the respiratory substrate, but this was not a direct effect on the alternative oxidase. In contrast, added GMP was a strong activator of fungal alternative oxidase activity. Analysis of plant and fungal alternative oxidase protein sequences revealed a unique domain of about 40 amino acids surrounding the regulatory Cys in the plant sequences that is not present in the fungal sequences. This domain may be where dimerization of the plant enzymes occurs. In contrast to plant enzymes, the fungal alternative oxidases studied here are monomeric and their activities are independent of α-keto acids.

Role of quaternary structure in the stability of dimeric proteins: the case of ascorbate oxidase.

Equilibrium denaturation experiments have been performed in order to study the dissociation into monomers and unfolding of the dimeric copper-containing enzyme ascorbate oxidase by urea and guanidine hydrochloride. The process has been followed by fluorescence intensity and anisotropy, by optical density, and by circular dichroism as a function of denaturant concentration. The noncoincidence of the unfolding curves obtained by different techniques suggests that a multiphasic process is occurring. The study of enzymatic activity and aromatic circular dichroism as a function of denaturant concentration shows that the first transition involves a change in the protein tertiary structure which is accompanied by the loss of biological function. Gel electrophoresis, ultracentrifugation, and protein dilution experiments demonstrate that a large fraction of protein molecules is still dimeric during this first transition with a stability which is strictly dependent on the denaturant used. The free energy change from the native form to this intermediate species was estimated to be approximately 3.5 kcal/mol. The binding of 1-anilino-8-naphthalenesulfonic acid to the partially unfolded, inactive ascorbate oxidase dimer also suggests a large conformational change accompanied by copper release, allowing the probe to penetrate deep inside the protein structure. Further denaturation to give a fully unfolded form is protein concentration dependent, suggesting that dissociation into monomers is occurring. The monomers appear to be very unstable. No evidence for structured intermediates was in fact detected in the last step of the denaturation process. A three-state model has been used to fit the fluorescence data, and the fractions of different species have been calculated as a function of denaturant concentration. The total free energy change of the unfolding transition using either urea or guanidine hydrochloride is rather small ( approximately 15-16 kcal/mol) and quite comparable to the value found for smaller proteins. The loss of secondary structure which occurs in the second part of the unfolding transition may be described by a simple two-state process which is characterized by a free energy change of 12-13 kcal/mol. These results suggest that the folding process of ascorbate oxidase follows a hierarchical model (Jaenicke, 1991). In this context, the assembly of monomers in a dimeric molecule plays a fundamental role by enhancing the protein stability and driving the final organization of the tertiary structure.

Kinetics and mechanism for the binding of HCN to cytochrome c oxidase

The kinetics of cyanide binding to cytochrome c oxidase were systematically studied as a function of [HCN], [oxidase], pH, ionic strength, temperature, type and concentration of solubilizing detergent, and monomer-dimer content of oxidase. On the basis of these results a minimum reaction mechanism is proposed in which the spectrally visible rapid and slow cyanide binding reactions are two consecutive first-order reactions, not parallel reactions with different conformers of cytochrome c oxidase. The fast reaction (k'obs) follows saturation type kinetics to form an HCN complex that subsequently undergoes a slow reaction (k'obs). The fast k'obs reaction is independent of ionic strength but is strongly dependent upon pH. Two pK values were evaluated from the bell-shaped rate versus pH profile; one is due to an ionizable group on the protein (pKa = 7.45), while the other is that of HCN (pKHCN = 9.15). Therefore, oxidase is reactive toward HCN only when the group on the protein is unprotonated. The slow k'obs reaction is not a reaction of oxidase with either CN- or HCN; in fact, the product formed by the fast k'obs reaction, the oxidase-HCN complex, still undergoes the slow k" process even if all of the excess KCN is removed. The apparent rate constant of the slower phase (k"obs) is independent of all the variations done in this study, and it probably corresponds to either a slow conformational change in the protein or a change in ligand coordination at one of the metal centers after HCN binds to the bimetallic center of oxidase. Based upon the bell-shaped pH dependence of the fast phase and the pH independence of the slow phase, the mechanism also predicts that a single conformer of cytochrome c oxidase can exhibit either monophasic or biphasic cyanide binding kinetics depending upon the pH. At either very low or very high pH, the two rates become comparable in magnitude, which makes the reaction appear to be monophasic even though both reactions still occur. The amount of monomeric or dimeric oxidase only slightly affects the magnitude of k'obs and k"obs values, and both processes are clearly present in both types of oxidase.

Electron microscopy of cytochrome c oxidase crystals: labeling of subunit III with a monomaleimide undecagold cluster compound.

Two-dimensional crystals of beef heart mitochondrial cytochrome c oxidase dimers were labeled at Cys-115 of subunit III with a monomaleimide derivative of an undecagold cluster compound. The binding site of the gold cluster compound and hence the site of subunit III were identified by image processing of cryoelectron micrographs of the crystals preserved in a mixture of glucose and uranyl acetate. The shape of the cytochrome oxidase dimer can be approximated as a parallelogram which is 44 by 82 A with an included angle of 80 degrees oriented with its long dimension along the a axis of the crystal. Labeling of subunit III was confirmed by a shift in the mobility of approximately 50% of subunit III molecules upon electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate. Averaged images of undecagold cluster labeled crystals and of unlabeled crystals were calculated; each image represents an average of approximately 17,000 molecules of either labeled or unlabeled cytochrome oxidase. On the basis of a statistical analysis of the differences between the two images, the gold cluster binds along a line 30 degrees from the a axis and 29 A from the center of the dimer. This result is interpreted in the context of other structural studies including the site of cytochrome c binding which Frey and Murray found to be near the a axis and 18 A from the center of the dimer [Frey, T. G., & Murray, J. M. (1994) J. Mol. Biol. 237, 275-297].(ABSTRACT TRUNCATED AT 250 WORDS)

Electron Microscopy of Cytochrome c Oxidase Crystals : Monomer-Dimer Relationship and Cytochrome c Binding Site

Cytochrome c oxidase was isolated from beef heart mitochondria by detergent extraction yielding two different crystal forms. Extraction with Triton detergents produced vesicular crystals with two-dimensional crystalline arrays of cytochrome c oxidase dimers while extraction with sodium deoxycholate produced crystalline sheets of cytochrome c oxidase monomers. The structures of both crystal forms were determined in two-dimensional projection along an axis normal to the plane of the membrane by cryoelectron microscopy of crystals embedded in vitreous ice (frozen-hydrated). The projection structures of unstained frozen hydrated monomers and of dimers are similar to the structures of the crystals in negative stain. The molecular outline of dimers can be approximated by a parallelogram 44 Å by 82 Å with an included angle of 80°. Monomers are less regular consisting of two large domains with a smaller domain at one end and a total length of approximately 82 Å. Comparison of the two structures reveals the orientation of cytochrome c oxidase monomers within dimers, an orientation which is different from earlier models of monomer-monomer interaction, and suggests a very close interaction between monomers when they associate to form dimers. The crystalline sheets of cytochrome c oxidase monomers bind tightly the small peripheral membrane protein substrate, cytochrome c , and this binding accentuates a tendency of these crystals to stack upon one another. Images of crystals of the cytochrome c oxidase/cytochrome c complex were analyzed by crosscorrelation analysis versus the monomer crystal image. Two types of two-layer crystals have been identified. Both types have one layer rotated by 180° with respect to the other, but they differ in the shifts of origin along crystal axes of the two layers. Difference images formed by subtracting simulated multilayered crystal images (which have no bound cytochrome c) from the complex crystals (cytochrome c oxidase plus cytochrome c) contain one positive difference peak for each cytochrome oxidase monomer within a unit cell. Comparison of the difference peak loci among the different crystal forms is interpreted based upon a consensus cytochrome c binding site in the single layer cytochrome oxidase monomer crystal image.

Cytochrome c oxidase: structural studies by electron microscopy of two-dimensional crystals.

Cytochrome c oxidase is a complex integral membrane protein consisting of 13 different polypeptide chains and four metal centers having a total molecular weight of approximately 200,000 daltons. It can be isolated in two 2-dimensional crystalline forms differing in aggregation state of the enzyme. One crystal form consists of cytochrome oxidase dimers (approximately 400,000 daltons) embedded unidirectionally in the lipid bilayer of a collapsed vesicle while the other form consists of crystalline sheets of cytochrome oxidase monomers. Both crystal forms have been studied by electron microscopy during the past two decades, and this paper summarizes the results of early structural studies as well as more recent results applying techniques of cryoelectron microscopy and digital image processing. The structure of frozen-hydrated cytochrome oxidase dimers at 20 A resolution is discussed as well as the packing of monomers within dimers and the site of cytochrome c binding.

Detection of bovine heart mitochondrial cytochrome c oxidase dimers in Triton X-100 and phospholipid vesicles by chemical cross-linking.

Bovine heart cytochrome c oxidase is a multisubunit enzyme whose oligomeric state is dependent on its detergent or phospholipid environment. We have utilized the cleavable, heterobifunctional cross-linking reagent N-succinimidyl 3-[(4-azidophenyl)dithio]propionate (SADP) to detect cytochrome c oxidase dimers. Monomeric or dimeric enzyme dispersed in Triton X-100 (as assessed by sedimentation velocity measurements) was reacted with SADP. A unique intersubunit cross-link having an apparent molecular mass of 136 kDa was identified in the dimeric enzyme; this product was insensitive to limited proteolysis by trypsin and contained a cross-link between two adjacent monomers. Two-dimensional NaDodSO4-PAGE (the second dimension containing beta-mercaptoethanol to cleave the cross-linking reagent) indicated that subunit I was the major component of the dimer-specific cross-link. The dimer-specific cross-link created by SADP was observed in phospholipid vesicles [cardiolipin/phosphatidylcholine (1:20, w/w)] containing dimeric (2 microM heme aa3) enzyme; a low yield of dimer-specific cross-link was observed in liposomes containing 6 microM (heme aa3) monomeric enzyme. The 136-kDa cross-link was not observed in liposomes containing 2 microM (heme aa3) monomeric enzyme. These results indicate that subunit I from each monomer may provide one site of interaction between monomers in the dimeric form of the enzyme and that cytochrome c oxidase monomers may reassociate to form dimeric complexes in phospholipid vesicles.

Electron cryo-microscopic analysis of crystalline cytochrome oxidase

The structure of cytochrome oxidase from beef heart mitochondria has been analysed by cryo-electron microscopy of vesicle crystals of the space group p22(1)2(1), with cell dimensions a = 102 A, b = 123 A, gamma = 90 degrees. Several methods of specimen preparation were applied to the vesicular two-dimensional crystals in the electron microscope, to ensure that the structure was preserved to the maximum resolution. The two most informative density maps were from specimens embedded in ice and from negative staining in a 1:1 mixture of glucose and uranyl acetate. The three-dimensional structure of the ice-embedded molecule shows a single, well resolved, but convoluted density, which represents in size and shape one cytochrome oxidase dimer. At the bottom of the molecule, a substantial part of the protein is embedded in the lipid bilayer of the vesicle. The molecule then extends upwards, out of the bilayer, into the internal space within the vesicle. Here, the structure first passes through a region within the molecule containing a hollow cavity that lies roughly at the centre of mass of the dimer, and then branches into two well-resolved halves at some distance from the membrane. The negatively stained structure, in contrast, shows a stain-excluding region in the centre of the vesicle at the level of the cavity in the ice-embedded structure, but otherwise has a similar overall external shape. In addition, there is a small rotation of the whole molecule by approximately 25 degrees relative to the orientation of ice-embedded specimens. We interpret these differences to mean that the central cavity seen in the ice-embedded structure is too small to allow the stain to penetrate during the drying process and that the drying process causes the rotation. The structures described here are consistent with one another and allow an interpretation at higher resolution than from previous work.

Ionic-strength-dependence of the oxidation of native and pyridoxal 5′-phosphate-modified cytochromes c by cytochrome c oxidase

The ionic-strength-dependences of the rate constants (log k plotted versus square root of 1) for oxidation of native and pyridoxal 5'-phosphate-modified cytochromes c by three different preparations of cytochrome c oxidase have complex non-linear character, which may be explained on the basis of present knowledge of the structure of the oxidase and the monomer-dimer equilibrium of the enzyme. The wave-type curve (with a minimum and a maximum) for oxidation of native cytochrome c by purified cytochrome c oxidase depleted of phospholipids may reflect consecutively inhibition of oxidase monomers (initial descending part), competition between this inhibition and dimer formation, resulting in increased activity (second part with positive slope), and finally inhibition of oxidase dimers (last descending part of the curve). The dependence of oxidation of native cytochrome c by cytochrome c oxidase reconstituted into phospholipid vesicles is a curve with a maximum, without the initial descending part described above. This may reflect the lack of pure monomers in the vesicles, where equilibrium is shifted to dimers even at low ionic strength. Subunit-III-depleted cytochrome c oxidase does not exhibit the maximum seen with the other two enzyme preparations. This may mean that removal of subunit III hinders dimer formation. The charge interactions of each of the cytochromes c (native or modified) with the three cytochrome c oxidase preparations are similar, as judged by the similar slopes of the linear dependences at I values above the optimal one. This shows that subunit III and the phospholipid membrane do not seem to be involved in the specific charge interaction of cytochrome c oxidase with cytochrome c.

Monomerization of cytochrome oxidase may be essential for the removal of subunit III.

1. Incubation of cytochrome oxidase, under conditions used as initial steps in treatment to remove subunit III, causes at least partial monomerization of the enzyme. 2. The extent of removal of subunit III by anion-exchange fast protein liquid chromatography (FPLC) is much increased if the enzyme is fully monomerized before it is applied to the column. 3. Subunit III is incompletely removed by chymotrypsin treatment. A digestion product of subunit III migrating in SDS-PAGE like subunit IV, is detected with specific antibodies. The amount of this product is reduced when monomerization is increased by raising the detergent/protein ratio. 4. The results suggest that monomerization facilitates removal of subunit III and exposes it to further chymotrypsin digestion. We propose that subunit III is at least in part located in the junction between the monomers in the cytochrome oxidase dimer.

Topological studies of monomeric and dimeric cytochrome c oxidase and identification of the copper A site using a fluorescence probe.

Beef heart cytochrome c oxidase was labeled at a single sulfhydryl group by treatment with 5 mM N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate (1,5-I-AEDANS) at pH 8.0 for 4 h. Sodium dodecyl sulfate gel electrophoresis revealed that the enzyme was exclusively labeled at subunit III, presumably at Cys-115. The high affinity phase of the electron transfer reaction with horse cytochrome c was not affected by acetylamidoethyl-1-aminonaphthalene-5-sulfonate (AEDANS) labeling. Addition of horse cytochrome c to dimeric AEDANS-cytochrome c oxidase resulted in a 55% decrease in the AEDANS fluorescence due to the formation of a 1:1 complex between the two proteins. Forster energy transfer calculations indicated that the distance from the AEDANS label on subunit III to the heme group of cytochrome c was in the range 26-40 A. In contrast to the results with the dimeric enzyme, the fluorescence of monomeric AEDANS-cytochrome c oxidase was not quenched at all by binding horse heart cytochrome c, indicating that the AEDANS label on subunit III was at least 54 A from the heme group of cytochrome c. These results support a model in which the lysines surrounding the heme crevice of cytochrome c interact with carboxylates on subunit II of one monomer of the cytochrome c oxidase dimer and the back of the molecule is close to subunit III on the other monomer. In order to identify the cysteine residues that ligand copper A, a new procedure was developed to specifically remove copper A from cytochrome c oxidase by incubation with 2-mercaptoethanol followed by gel chromatography. Treatment of the copper A-depleted cytochrome c oxidase preparation with 1,5-I-AEDANS resulted in labeling sulfhydryl groups on subunit II as well as on subunit III. No additional subunits were labeled. This result indicates that the copper A binding site is located at cysteines 196 and/or 200 of subunit II and that removal of copper A exposes these residues for labeling by 1,5-I-AEDANS. Alternative copper A depletion methods involving incubation with bathocuproine sulfonate (Weintraub, S.T., and Wharton, D.C. (1981) J. Biol. Chem. 256, 1669-1676) or p-(hydroxymercuri)benzoate (Li, P.M., Gelles, J., Chan, S.I., Sullivan, R.J., and Scott, R.A. (1987) Biochemistry 26, 2091-2095) were also investigated. Treatment of these preparations with 1,5-I-AEDANS resulted in labeling cysteine residues on subunits II and III. However, additional sulfhydryl residues on other subunits were also labeled, preventing a definitive assignment of the location of copper A using these depletion procedures.

Separation, stability and kinetics of monomeric and dimeric bovine heart cytochrome c oxidase

The stability of monomeric and dimeric bovine heart cytochrome c oxidase in laurylmaltoside-containing buffers of high ionic strength allowed separation of the two forms by gel-filtration high-performance liquid chromatography (HPLC). A solution of the dimeric oxidase could be diluted without monomerisation. Both monomeric and dimeric cytochrome c oxidase showed biphasic steady-state kinetics when assayed spectrophotometrically at low ionic strength. Thus, the biphasic kinetics did not result from negative cooperativity between the two adjacent cytochrome c binding sites of the monomers constituting the dimeric oxidase. On polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS) a fraction of subunit III of the dimeric enzyme migrated as a dimer, a phenomenon not seen with the monomeric enzyme. This might suggest that in the dimeric oxidase subunit III lies on the contact surface between the protomers. If so, the presumably hydrophobic interaction between the two subunits III resisted dissociation by SDS to some extent. Addition of sufficient ascorbate and cytochrome c to the monomeric oxidase to allow a few turnovers induced slow dimerisation (on a time-scale of hours). This probably indicates that one of the transient forms arising upon reoxidation of the reduced enzyme is more easily converted to the dimeric state than the resting enzyme. Gel-filtration HPLC proved to be a useful step in small-scale purification of cytochrome c oxidase. In the presence of laurylmaltoside the monomeric oxidase eluted after the usual trace contaminants, the dimeric Complex III and the much larger Complex I. The procedure is fast and non-denaturing, although limited by the capacity of available columns.

Preparation of reconstituted cytochrome oxidase vesicles with defined trans-membrane protein orientations employing a cytochrome c affinity column

Reconstituted cytochrome oxidase systems in which the majority of the vesicles contain a single oxidase dimer can be prepared. It is shown that, when these are passed through a cytochrome c affinity column, only those vesicles oriented outwards (such that the active site is available to external cytochrome c) are bound to the support matrix. Protein-free vesicles and vesicles containing an inwardly oriented enzyme are eluted in the void volume. Subsequently, vesicles containing an outwardly oriented enzyme can be eluted from the column at high salt concentrations. This protocol has been used successfully to resolve vesicles of either oxidase orientation when the enzyme is reconstituted with a variety of lipid mixtures. The recovery of oxidase activity from the column ranged between 75 and 94%.