Tryptophan residues cytochrome

Munro, A. W., et al. (1992) Investigating the function of cytochrome P450 BM-3 a role for the phylogenetically conserved tryptophane residue. Biochem. Soc. Trans. 21, 66. 

Figure 9.1 shows the fluorescence spectrum of tryptophan residues of cytochrome b2 core dissolved in 6 M guanidine, pH 7, in the presence of 30 mM 2-mercaptoethanol. The emission peak is located at 357 nm, indicating that the protein is completely denatured (spectrum a). The fluorescence emission spectra of successive aliquots (0.55 /uM) of free tryptophan added to the protein solution are also shown (spectra b-g). 

Figure 9.2 shows the fluorescence intensity maximum as a function of added free tryptophan. Extrapolation of the plot at the x-axis yields a value equal to 2 /xM as the concentration of tryptophan in the cytochrome b2 core. Since the protein concentration in the cuvette is 1 fiM, the number of tryptophan residues in the cytochrome b2 core is... 

Figure 9.2 Determination of tryptophan residues concentration in the cytochrome b2 core.

The cytochrome b2 core from the yeast Hansenula anomala has a molecular mass of 14 kDa, and its sequence shows the presence of two tryptophan residues. Their fluorescence intensity decay can be adequately described by a sum of three exponentials. Lifetimes obtained from the fitting are equal to 0.054,0.529, and 2.042 ns, with fractional intensities equal to 0.922, 0.068, and 0.010. The mean fluorescence lifetime, r0, is 0.0473 ns. 

Figure 11.4 Steady-state fluorescence anisotropy vs. temperature/viscosity ratio for tryptophan residues of cytochrome b2 core. Data are obtained by thermal variations in the range 10-36°C.

Figure 14.7 Location of five tryptophan residues (shown in a ball-and-stick representation) and the heme prosthetic group (white ball and stick) in the heme domain of cytochrome P-450BM3-...

Figure 14.8 Plot of k2 against the distance of tryptophan residues from the heme in cytochrome P-450. The dotted lines in the figure show the crystallographic distances between tryptophan residues, and lines labeled with rx and show the calculated distances between the heme and different tryptophan residues using all possible values of tc2 (0.0-4.0). The point where these two lines intersect shows the k2 value for the particular residue and is indicated by the arrows. Source of figures 11.22 and 11.23 Khan, K.K., Mazumdar, S., Modi, S., Sutcliffe, M., Roberts, G.C.K. and Mitra, S. (1997). European Journal of Biochemistry, 244, 361-370.

A H-bonded triad Hisl 18-Asp237-Trp48 (Figures 4 and 5A) between the diiron centre of R2 and the surface of the protein was recognised already in the initial report of the crystal structure of R2 (Nordlund et al., 1990). It mimicked a His-Asp-Trp triad in cytochrome c peroxidase (Edwards et al., 1988) that connects the metal ion of the heme group with a tryptophan residue known to harbour a transient radical (Huyett et al., 1995). 

In the first step (equation 1), peroxide removes two electrons from the protein. One derives from iron and another from an organic group, R, giving the well-known compound 1. Compound 1 thus has two fewer electrons than the native enzyme. In most peroxidases, the oxidized organic group is the porphyrin leading to a porphyrin tt-cation radical. However, in cytochrome c peroxidase (CcP), a tryptophan residue rather than the porphyrin is oxidized. ... 

The procedure outlined below, based upon the description of Previero et al. (1967b) has been used successfully, with minor variations for the selective modification of tryptophan residues in lysozyme (Previero et al. 1967b), trypsin (Coletti-Previero et al. 1969), cytochrome c (Aviram and Schejter 1971), and thioredoxin (Holmgren 1972). 

The crystalline preparation of E. coli cytochrome 6-562 172,173) shows absorption peaks at 418 nm in the oxidized form and peaks at 427, 531.5, and 562 nm in the reduced form. The cytochrome does not combine with CO in the pH range of 3.0-10.5 but at a higher pH CO causes a small shift of the a maximum. It does not react with cyanide or azide. Its molecular weight is calculated to be 12,000, 11,900-12,700, and 11,954, based on the heme content, ultracentrifugal data, and amino acid analysis, respectively. The amino acid sequence of cytochrome 6-562 has been determined Wl). As shown in Table VII this protein is composed of 110 amino acid residues, and lacks CySH and tryptophan. The cytochrome molecule possesses only two histidines, both of which probable coordinate to heme. Therefore, the cytochrome may be said to contain minimal numbers of histidines which are necessary to keep protoheme in the cytochrome molecule. However, it is also possible that one of the two histidines, especially near the carboxyl terminus, does not par-... 

As summarized earlier, there is consensus with regard to the sequence of electron transfer in cytochrome oxidase. The Cua center is the initial acceptor of electrons from cytochrome c (k 3 x 10 M s ). This electron transfer depends crucially on a conserved tryptophan residue in subunit II ca. 5 A away from the Cua center. Then follows fast electron equilibration between Cua and the low-spin heme (kf 10 s kr 5 x 10 s, kf and kr denoting the... 

Stayton PS, Sligar SG (1991) Structural micro-heterogeneily of a tryptophan residue required for efficient biological electron transfer between putidaredoxin and cytochrome P-450cam. Biochemistry 30 1845-1851... 

A molecule of horse-heart cytochrome c contains only one tryptophan residue. When the heme group is removed, to form apo-cytochrome c, the polypeptide chain is unable to assume a globular conformation, and exists as a random coil, with the tryptophan largely exposed to the solvent (Stellwagen e., 1972). The... 

Fourier transform spectra, were able to identify the resonances of Cs and C9 of tryptophan residues in horse heart cytochrome c and hen egg white lysozyme. 

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