Cytochrome c oxidase preparation

Specific Activities of Cytochrome c Oxidase Preparations from Different Isolation Procedures... 

Einarsdottir, O., and Caughey, W. S., 1985, Bovine heart cytochrome c oxidase preparations contain high affinity binding sites for magnesium as well as for zinc, copper and heme iron, Biochem. Biophys. Res. Comm. 129 8409847. 

Recent advances in measuring the kinetics of the various electron-transfer steps in this system have been achieved by use of flash photolysis of ruthenated derivatives of cytochrome c (Ru-Cc) (17-19). In these studies [Ru(bpy)3]2+ is covalently bound to a surface residue at a site that does not interfere with the docking of cytochrome c to cytochrome c oxidase. Solutions are then prepared containing both Ru-Cc and cytochrome c oxidase, and the two proteins associate to form a 1 1 complex. Flash photolysis of the solution leads directly to the excitation of the RuII(bpy)3 site, which then reduces heme c very rapidly. This method thus provides a convenient means to observe the subsequent intracomplex electron transfer from heme c to cytochrome c oxidase and further stages in the process. 

Chemical perturbations can be a useful approach to this problem. In laccase, selective replacement of the type 1 coppo- with mercury has allowed the type 1 site in laccase to be characterized stmcturally, even in the presence of three other copper atoms (27). Likewise, Simolo et al. have studied independently the a and 3 subunits in hemoglobin by preparation of the (a-M)2(P-M )2 derivaties, where M and M are different metals (28). An alternative to replacement is metal removal, for example Cu in cytochrome-c oxidase (29) or the T3 Ci in laccase (30). The concern with all of these approaches is to establish that the modified protein has the same metal-site structures as the native protein. 

Many group 13 compounds have been prepared with porphyrins. The majority of these compounds were created to serve as models for the active sites of enzymes, such as cytochrome c oxidase. Additionally, the gallium compounds are more robust than their iron counterparts. To a much lesser extent the Por-AlX compounds are used as oxirane polymerization catalysts [8]. 

Complex IV (cytochrome c oxidase) activity is measured by following at 550 nm (e 19,100-MAcnr1 isosbestic point 540 nm) the oxidation of cytochrome c (Fig. 3.8.5). It could be calculated as a first-order rate constant, or by estimating the pseudolinear initial rate of the reaction. Noticeably as a check, this initial rate should represent about double the rate measurable when 50% of the added cytochrome c has been oxidized (Fig. 3.8.5). Indeed, the affinity constant (Km) for reduced cytochrome c is about equivalent to the K, for oxidized cytochrome c. Reduced cytochrome c is easily prepared by adding a few crystals of dithionite to a solution of oxidized cytochrome c. After the immediate color change (from deep red to light orange-red), the solution should be carefully stirred to eliminate any trace of dithionite and should be totally odorless. 

It is well known that the O2 reduction site of bovine heart cytochrome c oxidase in the fuUy oxidized state exhibits variable reactivity to cyanide and ferrocytochrome c, which is dependent on the method of purihcation (Moody, 1996). Some preparations react with cyanide extremely slowly at an almost immeasurable rate and are known as the slow form. Other preparations, which react at a half-Ufe of about 30 s, are known as the fast form (Brandt et al., 1989). Electronic absorption spectra of the slow-and fast-form preparations exhibit Soret bands at 418 and 424 nm, respectively. The two forms often coexist in a single preparation (Baker et al., 1987). Both forms exhibit an identical visible-Soret spectrum in the fully reduced state. The slow-form preparation can be converted to the fast form by dithionite reduction followed by reoxidation with O2. The fast form thus obtained returns to the slow form spontaneously at a rate much slower than the enzymatic turnover rate. Thus, the slow form is unlikely to be involved in the enzymatic turnover (Antoniniei a/., 1977). It should be noted that no clear experimental evidence has been reported for direct involvement of the fast form in the enzyme turnover, although its direct involvement has been widely accepted. The third species of the fully oxidized O2 reduction site, which appears in the partially reduced enzyme, reacts with cyanide 10 —10 times more rapidly than the fast form (Jones et al., 1984). In the absence of a reducing system, no interconversion is detectable between the slow and the fast forms (Brandt et al., 1989). Thus, the heterogeneity is expected to inhibit the crystallization of this enzyme. In fact, the enzyme preparations providing crystals showing X-ray diffraction at atomic resolution are the fast form preparation. 

In order to elucidate the reaction mechanism of cytochrome c oxidase, the complete structure of the enzyme must be determined. The first step in this process is the complete determination of its composition. X-ray crystallographic analysis at high resolution was required in addition to chemical analysis for crystalline enzyme preparation. 

Fig. 9. A reductive titration of the crystalline bovine heart cytochrome c oxidase with dithionite. Absolute spectra for each oxidation state are shown for the Soret (A) and visible (B) regions. The difference spectra against the spectrum in the fully reduced state are given for the near-infrared region (C). The insets show titration curves against the electron equivalent per enzyme. The reaction mixture contained 7.5 jlM bovine heart cytochrome c oxidase in 0.1 M sodium phosphate buffer, pH 7.4. The enzyme preparation was stabilized with a synthetic non-ionic detergent, CH3(CH2)ii(0CH2CH2)80H. The light path was 1 cm.

The redox properties of cytochrome c oxidase have been investigated both by anaerobic reductive titrations 159) and by potentiometric titrations (160). Since measurements of the latter kind are, at least in principle, able to provide absolute potential values, they have been favored in recent studies. The inconsistencies found in the early work (161-163) may have resulted from the lack of equilibrium conditions in some cases, from differences in the preparations, or simply from some incorrect interpretations of data. The importance of establishing that equilibrium conditions are attained has recently been recognized (107, 124,1 5), but identical sets of measurements on the various types of preparations have yet to be reported. 

Several heterodinuclear Fe-Cu or Co-Cu complexes that closely resemble the native enzyme active sites have recently been prepared to elucidate the catalytic mechanism of cytochrome c oxidases [231-245], The use of a covalently attached axial ligand seems essential to achieve efficient electroreduction of O2 to H2O [235, 238, 241-243], The closest structural analogs of the heme as/Cus active site of cytochrome c oxidases reported so far are Fe-Cu complexes (1 and 2) in which the Cu coordination site is provided by three imidazole ligands [242], These biomimetic model complexes afford clean electroreduction of O2 to H2O over a wide range of pH with no leakage of H2O2 [243],... 

These findings are consistent with impaired fatty-acid oxidation reduced mitochondrial entry of long-chain acylcarnitine esters due to inhibition of the transport protein (carnitine palmityl transferase 1) and failure of the respiratory chain at complex II. Another previously reported abnormality of the respiratory chain in propofol-infusion syndrome is a reduction in cytochrome C oxidase activity, with reduced complex IV activity and a reduced cytochrome oxidase ratio of 0.004. Propofol can also impair the mitochondrial electron transport system in isolated heart preparations. 

Cytochrome c oxidase of N. europaea was called cytochrome au as it shows the a peak at 595 nm (Erickson et al., 1972). However, the electrophoretically homogeneous preparation of the oxidase has two heme A molecules and two copper atoms (Cua and Cub) in the molecule, and one of the two heme A molecules reacts with... 

Frosolono and Pawlowski (1977) studied biochemical changes in various lung fractions prepared from rats exposed to phosgene at concentrations near to or above the LCtso. A number of enzymes showed decreased activity in all fractions these included p-nitrophenyl phosphatase, cytochrome c oxidase, ATPase and lactate dehydrogenase (LDH). The serum LDH rose. It was suggested that either inhibition of enzyme activity or loss of enzyme from cells would account for these changes. The data available did not allow... 

An important aspect of the preparation and isolation of subcellular particles from brain regions is the criteria by which purity is assessed. Electron microscopy of the various subcellular fractions can provide among the best pieces of evidence for the presence in the preparation of the organelles or subcellular fragments of interest. However, a number of biochemical markers (usually enzymes) that have been established to be present in certain fractions can also be assayed to demonstrate the enrichment of the organelle of interest. For instance, acetylcholinesterase is a common marker for synap-tosomes dopamine-P-hydroxylase is a marker for catecholamine storage vesicles within the synaptosome and cytochrome c oxidase is a marker for mitochondria. Most of the enzymatic markers can be assayed routinely. 

Figure 38 The redox-active cofactors of cytochrome c oxidase and the D and K proton transfer pathways (PDB-code 1M56), the red spheres are water moiecuies resoived in the X-ray crystai structures prepared with PyMOL (W. L. DeLano, Paio Alto, 2003).

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