Cytochrome subunit composition

Subunit Composition of Bovine Heart Cytochrome be I Compiex... 

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). 

These are similar to the mitochondrial enzyme, apart from the simpler subunit composition. EXAFS studies on the oxidases from Paracoccus denitrijicans and Thermus thermophilus have confirmed the metal centres to be similar to those in the mitochondrial enzyme. In the latter case, the ESR silent cytochrome Us in the reduced enzyme has been shown by the use of Mossbauer spectroscopy to involve five-coordinate, high-spin On addition of cyanide to the oxidized... 

The subunit composition determined by SDS/urea-PAGE is given in Table I. Immunoblot analysis identifies two subunits of 18.5 kDa and the 9.5 kDa subunit as the psaD. psaF and psaC gene products, coding for the ferredoxin-, the cytochrome c-and the FeSA/FeSe-binding protein, respectively (not shown 5). While the two big subunits may be attributed to the psaA and osaB gene product (5), the identity of the other four small subunits is unknown. 

Application of the impure bef complex to the hydroxyapatite column yielded a similar profile as in Fig. 2b, however with a predominant cyt.bef-peak (not shown). The four subunits of about 38, 24, 19 and 15 kDa (Table I) are likely to be cytochrome f- and cytochrome be-binding subunit, the Rieske Fe-S-protein and subunit IV, respectively, consistent with the subunit composition of other cyanobacterial cyt.bef-complexes. The presence of b-type and c-type cytochromes in a ratio of 2 1 was further confirmed by reduced-minus-oxidised difference spectra (not shown). 

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.

The subunit composition of cytochrome b was analyzed by means of polyacrylamide gel electrophoresis in a dodecylsulfate medium. In contrast to bile acids, the detergent, dodecylsulfate, caused cytochrome b to dissociate into polypeptide(s) and heme. The protein moiety of cytochrome b migrated as one band (Fig. 15). By calibrating the electrophoretic mobility with proteins of known molecular weight, an apparent molecular weight of 30,000 could be attributed to this protein band. 

A second example of a membrane-bound arsenate reductase was isolated from Sulfurospirillum barnesii and was determined to be a aiPiyi-heterotrimic enzyme complex (Newman et al. 1998). The enzyme has a composite molecular mass of 100kDa, and a-, P-, and y-subunits have masses of 65, 31, and 22, respectively. This enzyme couples the reduction of As(V) to As(III) by oxidation of methyl viologen, with an apparent Kra of 0.2 mM. Preliminary compositional analysis suggests that iron-sulfur and molybdenum prosthetic groups are present. Associated with the membrane of S. barnesii is a h-type cytochrome, and the arsenate reductase is proposed to be linked to the electron-transport system of the plasma membrane. 

There are four different types of nitrite reductases the copper-containing protein Copper Enzymes in Denitrification and cytochrome cd perform a one-electron rednetion of nitrite to nitric oxide, and are involved in denitrification " the siroheme-containing protein and the cytochrome c ititrite reductase (cNiR) both perform the complete, six-electron reduction, of nitrite to ammonia. The cNiR is present in the y, 5 or e-subclasses of proteobacteria, and is encoded by the nrf operon (nitrite reduction with /ormate), which has different gene composition in the different classes of bacteria, having in common only the gene for the catalytic subunit, ntfA. 

The chemical composition of the highly purified reaction-center complex from several photosynthetic bacteria is now well established. In addition to protein subunits designated as L (light), M (medium) and H (heavy), some reaction centers also contain a c-type cytochrome (C) subunit. The chemical composition of reaction-center complexes of several of these bacteria is shown in Table 1 ... 

Reduction of plastocyanin by cytochrome / would presumably involve some direct interaction between them. Cyt/will be described in detail in Chapter 35, but some relevant information on the composition and structure of Cyt / is given here to provide a perspective on the PC/Cyt / interaction. Cyt /, a subunit of the Cyt complex, is a c-type cytochrome of 33 kDa molecular mass with an in situ redox potential of -370 mV. The amino-acid composition has been determined for Cyt/from several species, that for turnip being shown in Fig. 11. Turnip Cyt/, containing 252 amino-acid residues, was crystallized and its three-dimensional structure determined in 1994 by Martinez, Huang, Szczepaniak, Cramer and Smith " at 2.3 A resolution, now refined to 1.96 A (see Chapter 35). 

Fig. 2. (A) A model for b/in chloroplast thylakoid membrane (B) Topological arrangement of the four subunits of the purified Cyt b/complex (C) A densitometric scan of an SDS-PAGE gel for b/ (B) from Hauska, Schiitz and Biittner (1996) The cytochrome b/ complex - composition, structure and function. In DR Ort and CF Yocum (eds) Oxygenic Photosynthesis - The Light Reactions, p 384. Kluwer (C) from Black, Widger and Cramer (1987) Large-scale purification of active cytochrome b f complex from spinach chloroplasts. Arch Biochem Biophys 252 657.

Color Plate 15. Composite structure of vertebrate mitochondrial Cyt bCy The dark horizontal band indicates the probable membrane position. The left and right figures are related by a rotation of 90 about the membrane normal. Color codes for subunits listed in the figure. (Courtesy of Dr. E. A. Berry and Dr. A. R. Crofts. Reference source EA Berry, M Guergova-Kuras, L-S Huang and AR Crofts (2000) Structure and function of cytochrome be complexes. Annu Rev Biochemistry (69 1024). [See Chapter 35, Sections I.D. and III.C.]... 

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