What are the most accurate extinction coefficients for determining the concentration of the bc1 complex or its individual chromophores? Are there significant differences between species?
Not important for many purposes, but it is if you are looking at stoichiometry in supercomplexes, studying the shape of inhibitor titration curves, counting the quinones, or quibbling over who has the highest turnover number!
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Probably the best extinction coefficients are still those from van Gelder's review in Methods in Enzymology Volume 53, p125.
The species does make a difference- not only in wavelength but also in shape of the curves and so presumably extinction coefficient. Purple bacteria look very like bovine, but with L-max 560 instead of 562-3. Fish, birds, and plants look distinctly different (Degli-Esposti, Berry), with broader b(H) alpha peak. In the potato complex L-max is actually 558, which is the 558 shoulder of b-low riding on a flat plateau resulting from summation of cyt c1, b(H), and 566 peak of b(L). Potato and chicken bc1 have pretty typical b(L) wit 566/558 peaks. One old spectrum from Palmer's lab shows yeast b(L) with such a split spectrum, but we have not been able to show it. Trypanosome bc1 has a very odd spectrum for cyt b (of course cyt c1 is shifted because of the single thioether linkage). And going farther afield, Thermus bc1 (Ludwig) and cyt bc6f are spectrally quite different from the bovine complex.
For bovine cyt bc1, the most commonly used ext coeff for quantitating the whole complex is the reduced-oxidized cyt b dual-wavelength difference at 563-577 (alpha peak minus red-side trough). Berden and Slater give this as 28.0 /mM based on pyridine hemochrome analysis, as compared to 28.5 from earlier work by Zaug and Rieske using the same method. Antimycin titration gave a lower value of 25.6, but we believe the extinction coefficient used for antimycin was too low (see below), so the 28 - 28.5 values is probably best.
This can be applied to a dithionite-reduced vs hydroquinone- or ascorbate reduced preparation, or since the contribution of cyt c1 at this wavelength pair is quite small, to fully reduced vs fully oxidized complex.
There has been some confusion whether this value should be taken as per monomer of bc1 (i.e. per two hemes) or per heme. Remember in those days the complex was believed to contain two monoheme cyt b. In any case Berden and Slater are quite clear: "Based on the assumption that the bc1 segment of the respiratory chain contains 2b:1c(1):1 antimycin binding site, (anti binding gives) extinction coefficients of b and c1. These are 25.6 and 20.1 mM-1 cm-1." So clearly the extinction coefficient of the complex wold be twice that of cyt b, or 51 based on antimycin, 56 from pyridine hemochromes.
The above-mentioned value 20.1 for cyt c1 from antimycin binding is also a reduced - oxidized value, at 553-539 (alpha peak minus blue-side trough). van Gelder titrated cyt c1 with NADH (pms as mediator) and got an extinction coefficient of 20.9 at 552-540. (His M.E. review confirms that the 25.6 value for cyt b is "on a heme basis".)
van Gelder's value for the extinction coefficient of cyt c1 was from very carefully done experiments using three different methods that agreed well. However it was done using purified cyt c1, and there is some indication (ref needed) that heterogeneous environment in the cyt c1 micelle leads to broadening of the peaks, in which case the in-situ extinction coefficient would be larger. In fact it was shown that the integrated area of the reduced alpha peak was similar to that of soluble cyt c, even though the peak height was smaller. Going back to the Berden& Slater reference, the ratio between the diff ext coef for cyt b (peak-red trough) and cyt c1 (peak minus blue-side trough) is 25.6/20.1 or 1.27. Thus if we use an extinction coefficient of 28.5 for cyt b, the value for cyt c1 should be 22.4; 28.0 -> 22.0. This is still below that of cyt c (25. at peak - blue-side trough) given by van Gelder
I put digital spectra of the cytochromes of bc1, our best approximation which we are using to quantitate the cytochromes, in an MS Excel spreadsheet at http://sb20.lbl.gov/spectra/bovine_bc1.xls.
These are based on singular-value decomposition of a nonpotentiometric titration to get a basis, potentiometric titration to assign the portion of each basis component to each titrating species. Finally the spectra were put on an absolute basis by fitting fully reduced minus oxidized spectra of intact purified complex for which the heme content was known by pyr hemochrome analysis as a linear combination of the resulting spectra (method is described in our 91 JBC paper on potato bc1).
Shinkarev and coworkers have evidence that there is redox interaction between the two hemes B and their intrinsic Em's are much closer than indicated by potentiometric titration. In this case the first redox step, which I call b(H), is really going from oxidized to the 1-electron state, where the 1 electron is shared something like 80:20 between b(H) and b(L); i.e. the difference spectrum contains some b(L). The second redox transition which I call b(L) is then going from the 1-electron to 2-electron state, i.e. the rest of b(H) and b(L) go reduced, so this spectrum contains some b(H).
Antimycin ext. coeff.: Berden and Slater used 4.8 mM-1 from Strong et al (reported as logE320 = 3.68). However Birch et al. reported E319 = 3.78, corresponding to a millimolar extinction coefficient of 6.03. Antimycin dissociates slightly in ethanol solution due to basic impurities in the solvent. Therefore it is necessary to acidify dilute solutions before taking a spectrum. This suggested acidometric titration as a good way to determine concentration/extinction coefficient of the chromophore, which is the same in the different forms (Antimycin A1, A2, A3). Preliminary results indicate that the extinction coefficient at 319-320 is significantly greater than 4.8.
ref's:
1. van Gelder, B. F. (1978). Optical properties of cytochromes from beef heart mitochondria, submitochondrial vesicles, and derived preparations. Methods Enzymol 53, 125-8.
2. Berden, J. A. & Slater, E. C. (1970). The reaction of antimycin with a cytochrome b preparation active in reconstitution of the respiratory chain. Biochim Biophys Acta 216, 237-49.
3. Berry, E. A., Huang, L. S. & DeRose, V. J. (1991). Ubiquinol-cytochrome c oxidoreductase of higher plants. Isolation and characterization of the bc1 complex from potato tuber mitochondria. J Biol Chem 266, 9064-77.
4. Strong, F. M., Dickie, J. P., Loomans, M. E., Tamalen, v. & Dewey, R. S. (1960). JACS 82, 1513.
5. Birch, A. J., Cameron, D. W., Harada, Y. & Rickards, R. W. (1961). The structure of the antimycin-A complex. J. Chem Soc. 1961, 889-895.
6. Shinkarev, V. P., Crofts, A. R. & Wraight, C. A. (2001). The electric field generated by photosynthetic reaction center induces rapid reversed electron transfer in the bc1 complex. Biochemistry 40, 12584-90.
February 14, 2007 9:53:00 PM PST
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I took another look at Palmer's yeast bL spectrum- in fact the split is much less obvious than in vertebrates or bacteria- this is Figure 6 of Siedow and Palmer '78 - the peak of "b566" is actually at 563.5-564, making the shoulder at 558 much less noticeable. Even a little bit of bH (at 562.5) could completely mask the split. Perhaps we just haven't seen a sufficiently pure spectrum of bL yet to notice the split peak.
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