Cytochrome b2 core

Chromophores free in solution and bound to macromolecules do not display identical s values and absorption peaks. For example, free hemin absorbs at 390 nm. However, in the cytochrome b2 core extracted from the yeast Hansenula anomala, the absorption maximum of heme is located at 412 nm with a molar extinction coefficient equal to 120 mM-1 cm-1 (Albani 1985). In the same way, protoporphyrin IX dissolved in 0.1 N NaOH absorbs at 510 nm, whereas when it is bound to apohemoglobin, it absorbs in the Soret band at around 400 nm. 

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 9.1 Titration of 1 /xM cytochrome b2 core in 6 M guanidine with aliquots of 5.5 fiM of L-Trp.

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. 

The main fluorescence lifetime (r=54 ps) and its important fractional intensity (fi = 92%) indicate that an important energy transfer occurs between Trp residues and heme. In an attempt to measure rotational correlation time of the protein, we have measured anisotropy of cytochrome b2 core Trp residues at different temperatures. Results are described with the classical Perrin plot (1 /A as a function of T/p) (Figure 11.4). 

The data yield a rotational correlation time equal to 38 ps instead of 5.9 ns calculated theoretically for the cytochrome b2 core, with an extrapolated value A(o) of 0.208, lower than that (0.265) usually found for Trp residues at Xex = 300 nm at —45°C. The fact that the extrapolated anisotropy is lower than the limiting anisotropy means that the system is depolarized as a result of global and local motions within the protein. In this case, the value of the apparent rotational correlation time ( a) calculated from the Perrin plot is lower than the global rotational time of the protein ( bp). However, the fact that 4>a is 1000 times lower than 4>p indicates that a third process different than the global and local rotations is... 

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.

Electron transfer between electron donor and acceptor located in two different proteins or within the same protein induces a spectral modification in the absorption of both redox centers and thus kinetics parameters and electron transfer data can be studied with absorption. Figure 1.23 displays the absorption spectra of oxidized (a) and reduced (b) forms of cytochrome b2 core extracted from the yeast Hansenula anomala. Reduction of cytochrome c with cytochrome b2 core is followed at the isobestic point of cytochrome b2 equal to 416.5 nm. At this wavelength, only absorption of cytochrome c increases upon reduction. 

Figures 1.28 and 1.29 display respectively the variation of the second-order rate constant with ionic strength for flavocytochrome b2 cyt.c and cytochrome b2 core cyt.c electron transfer, illustrated by the classical Debye-Hiickel plot defined by log k+ versus the square root of ionic strength ...

A conclusion that can be drawn from these results is that the reactivity of cytochrome b2 core towards cytochrome c is not the same. The reason for this could be the less stable complex. Therefore, one role of the flavodehydrogenase is to stabilize the flavocytochrome b2 - cyt.c complex. It is important to mention that the of the flavodehydrogenase - cyt.c complex is equal to 0.1 pM(Albani, 1993). 

Figure 1.29, lonic strength dependence of the bimolecular rate constant, for the reduction of cytochrome c by cytochrome b2 core. Temperature = 5 C. Source CapeilUre-Blandin. C. and J. Albanu 1987. Biochem. J. 245,159-165. Authorization of reprint accorded by Portland Press. 

In presence of TNS, reduction of cytochrome c occurs in the range of minutes instead of seconds, as it is the case for the reduction with flavocytochrome b2 or isolated cytochrome b2 core. Thus, the relative distance and orientation of the FMN, TNS and heme planes induces an electron transfer pathway different from that known for the cytochrome b2 - cytochrome c electron transfer. This clearly shows the importance of cytochrome b2 core in the electron transfer to cytochrome c. 

All these studies indicate that electron transfer within the flavocytochrome -cytochrome c complex is dependent upon a number of factors such as the distance between donor (cytochrome b2 core or TNS) and acceptor (cytochrome c). their relative orientation, their chemical nature and the structure of the protein medium involved in the electron transfer. 

Usually, when studying protein-protein interaction, fluorescence variation of the probe can indicate the type of the interaction. For example, one can find out whether the interaction between the two proteins is cooperative or not, etc... Two examples to illustrate this, we used 2,p-toluidinylnaphthalene-6-sulfonate (TNS) as a fluorescent probe to study the interaction between cytochrome c and cytochrome hj core and to follow the binding of cytochrome b2 core on flavodehydrogenase. The three proteins are extracted from the yeast Hansenula anomala (see chapter 1, paragraph 6Q. 

Also, this means that the cytochrome c - cytochrome b2 core interaction is weaker than the interaction observed between cytochrome c with the two other proteins. Electrostatic interaction would pre-orient the proteins before any physical contact, facilitating the formation of a complex. Thus, when the interactions are weak, the pre -orientation is not adequate at every collision and the probability of formation of a complex is weak... 

Thus the Kd found at 100 mM ionic strength for the cytochrome c-apocytochrome b2 core case is higher than the Kd for the cytochrome c-cytochrome b2 core case in this ionic strength range. However, at 20 mM ionic strength, the effects of electrostatic... 

Albani, J. R., 1997, Interaction between cytochrome b2 core and flavodehydrogenase from the yeast Hansenula anomala. Photochem. Photobiol. 66, 72 - 75. 

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