Cytochrome oxidase composition

Amino acid sequence analysis and determination of subunit composition are painstaking but these steps are usually necessary before further structural investigations are undertaken. It should not be forgotten that chemical composition and amino acid sequencing provided a foundation for recent structure-function findings in the cytochrome oxidase field. The complete amino acid sequence and a successful prediction of the number of a-helices greatly contributed to the successful and rapid crystallographic analysis of bovine heart cytochrome c oxidase at 2.8 resolution, four years ago (Tsukihara et al., 1995 Tsukihara et al., 1996). 

Nagata Y, Yamanaka T, Okunuki K (1970) Amino acid composition and iV-terminus of Pseudomonas cytochrome oxidase (= Pseudomonas aeruginosa nitrite reductase). Biochim Biophys Acta 221 668-671... 

Composites of gold and PANl on a glassy carbon electrode were also used by Xiang et al. [52] for the development of a glucose biosensor. They used a bienzy-matic approach where glucose was oxidized thereby producing H2O2, which was reduced by cytochrome oxidase c, absorbed on the surface of the nanocomposites. This sensor enabled a direct electron transfer to the electrode material. The sensor has a detection limit of 0.01 mM. 

PANI was also used in combination with carbon nanotubes to fabricate a sensor with cytochrome oxidase as bioelement [57]. This systan could be used for different kinds of sensing events based on the enzymatic reduction of H202- The sensitivity of a biosensor based on a PANI film on a gold electrode was improved by using a composite of PANI with pol) yrrole nanoparticles [58]. The sensor was used to detect glutamate in food products with an enzymatic reaction of the covalently bound glutamate oxidase. 

Several comprehensive papers have recently appeared in which the four-electron reduction of molecular oxygen to water catalyzed by cytochrome oxidase has been discussed from various aspects, such as composition and properties of the enzyme and associated cytochromes, organization and behavior of the respiratory chain which contains the oxidase, and its relationship to oxidative phosphorylation (151, 295,464,673,707,782). There is now little to add to this literature... 

Fig. 3. Subunit composition of two types of cytochrome oxidase. Enzymes isolated and purified as described in the text, with the properties shown in Table II. Gel electrophoresis of dissociated proteins was as described by Fairbanks et al. on 10% gels at 6 mA per gel for 4 h staining was with Coomassie blue and scanning at 630 nm. (A) and (B) enzymes from beef heart (C) and (D) enzymes from yeast (A) and (C) type I (B) and (D) type II.

In order to define the translation site of the cytochrome b subunit(s) we proceeded as described for cytochrome oxidase (Table I) N. crassa was first labeled with [ C]leucine in the absence of inhibitors, and then with PHJleucine, either (1) in the absence of inhibitors, (2) in the presence of cycloheximide, (3) in the presence of chloramphenicol, or (4) during transitory presence of chloramphenicol. Purified cytochrome b was analyzed for and after gel filtration in bile acid-KCl solution and after gel electrophoresis in dodecylsulfate solution. It should be remembered that in the bile acid solution, the polypeptide and heme composition of cytochrome b remains intact, whereas in dodecylsulfate, the heme is dissociated from polypeptides. The results of these labeling experiments are shown in Table I and in Figs. 16 and 17. 

Analysis of the amino acid composition of the mitochondrially synthesized subunits of cytochrome oxidase of the rutamycin-sensitive ATP-ase (another membrane component which requires mitochondrial protein synthesis for some of its component polypeptides) and cytochrome b displayed a high proportion of nonpolar amino acid residues. The resulting unusually hydrophobic composition explains their insolubility in aqueous solutions. This hydrophobic character of the mitochondrially translated polypeptides may have relevance for speculations on the existence of mitochondrial protein synthesis. It has been suggested that their hydro-phobic properties make it necessary that these polypeptides be delivered to the inner mitochondrial membrane from the matrix side, since they cannot be transported through the cytoplasm and the intercristae space. 

The data presented in Table 3, which includes the amino acid composition of baker s yeast and Candida krusei cytochrome c for comparison, show that Ustilago and Neurospora cytochrome c contain the same number of total residues. In seven instances, the number of residues of a particular amino acid/mole are identical. Thus, even in the absence of a sequence for the Ustilago cytochrome it can be concluded that this protein, unlike the siderochromes, has suffered little alteration in the progression from the Ascomycetes to the Basidiomycetes. This can be ascribed to the varying function of the two types of molecules. Cytochrome c must fit into a relatively specific slot bounded by a reductase and an oxidase and it has hence evolved much more slowly than the more freely acting transport agents where the specificity constraints are less demanding. 

Several enzymes have been immobilized in sol-gel matrices effectively and employed in diverse applications. Urease, catalase, and adenylic acid deaminase were first encapsulated in sol-gel matrices [72], The encapsulated urease and catalase retained partial activity but adenylic acid deaminase completely lost its activity. After three decades considerable attention has been paid again towards the bioencapsulation using sol-gel glasses. Braun et al. [73] successfully encapsulated alkaline phosphatase in silica gel, which retained its activity up to 2 months (30% of initial) with improved thermal stability. Further Shtelzer et al. [58] sequestered trypsin within a binary sol-gel-derived composite using TEOS and PEG. Ellerby et al. [74] entrapped other proteins such as cytochrome c and Mb in TEOS sol-gel. Later several proteins such as Mb [8], hemoglobin (Hb) [56], cyt c [55, 75], bacteriorhodopsin (bR) [76], lactate oxidase [77], alkaline phosphatase (AP) [78], GOD [51], HRP [79], urease [80], superoxide dismutase [8], tyrosinase [81], acetylcholinesterase [82], etc. have been immobilized into different sol-gel matrices. Hitherto some reports have described the various aspects of sol-gel entrapped biomolecules such as conformation [50, 60], dynamics [12, 83], accessibility [46], reaction kinetics [50, 54], activity [7, 84], and stability [1, 80],... 

One of the cellobiose oxidoreductases present in S. pulverulentum has been characterised and named cellobiose oxidase (Ander and Eriksson, 1978). The enzyme contains both haem and flavin co factors and binds irreversibly to concanavalin A-Sepharose, suggesting that it is a glycoprotein. Cellobiose oxidase from S. pulverulentum has now been purified to homogeneity by Morpeth (1985). The carbohydrate and amino acid compositions of the enzyme have been determined. The enzyme contains FAD and cytochrome b prosthetic groups and is a monomer with an Mr of 74400 determined by sedimentation equilibrium. 

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. 

It has long been proposed that cytochrome c oxidase contains various phospholipids as intrinsic constituents and that two or three cardiolipins are essential for its catalytic activity (Robinson et al., 1980 Robinson, 1982 Sedlak and Robinson, 1999). However, the composition and structures of phospholipids in the enzyme are still matters of controversy. No experimental evidence has been obtained to discount the proposal that all phospholipids suggested are not intrinsic components of the enzyme. Thus, a careful phospholipid analysis of the crystalline enzyme and an X-ray structure determination are indispensable for the determination of the phospholipid composition. 

Phospholipid Composition op Cytochrome c Oxidase and Mitochondria from Bovine Heart... 

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