Mitochondrial Bioenergetics by Carlos M. Palmeira & António J. Moreno

Author:Carlos M. Palmeira & António J. Moreno
Language: eng
Format: epub
Publisher: Springer New York, New York, NY

1.6 Cytochromes

Complex III and Complex IV contain hemoproteins termed “cytochromes” which are readily detected by spectrophotometry. These were used as early as 1952 to measure mitochondrial redox changes in cells [15]. The measurements of the redox states of cytochromes depend upon the absorbance characteristics of the hemes in the cytochromes, which have more intense absorbance in the reduced forms (Fig. 1) [13]. In the visible range, the absorbance bands are termed α, β, and γ (Soret) absorbance bands. While the Soret band has the greatest extinction coefficients, it occurs at the shortest wavelength where there is the greatest light scatter in preparations containing mitochondria; consequently, measurements in this region can be more prone to error due to this light scatter. In addition, there is considerable overlap of signals from hemes b and c, and these can be further obscured by signals from myoglobin or hemoglobin [30]. Consequently, measurements in the Soret region are usually limited to cytochromes a 3. Cytochromes a and a 3 contain heme a, which is structurally distinct from heme b, present in hemoglobin, myoglobin, catalase, and b- and c-type cytochromes. Cytochromes a and a 3 also absorb light in the near-infrared region, and this has provided a means to measure oxidation in vivo [17]. Considerable spectral overlap occurs with the β bands, so most measurements are obtained with the α bands. Cytochromes b and c contain heme b (Fe-complexed protoporphyrin IX) but differ in absorbance because the heme is covalently bound in cytochromes c and c1 but not in b H or b L. Other minor differences in absorbance maxima occur due to the interactions with amino acids in the vicinity of the heme. Addition of ligands like KCN , which bind to the O2 site, can be used to discriminate cytochromes a and a 3. However, this is not useful for steady-state redox measurement because it blocks electron flow .

Complex III accepts two electrons from CoQ and transfers one to cytochrome c 1 and the other to the cytochrome b H and b L pair. The electron from cytochrome c 1 is then transferred to cytochrome c, while the electron from cytochrome b is transferred back to CoQ. The system is a proton pump, with energetics of proton pumping probably defined by steady-state potential of the donor ubiquinol/ubisemiquinone couple and cytochrome c 1. As above, changes in the steady-state E h values determine the energetics available for proton pumping. Consequently, interpretations of Complex III function in generation of ΔμH+, ROS generation or support of ATP production, are limited by changes in steady-state E h values which determine the available ΔE h.

Steady-state reduction of cytochromes b is measured by dual-wavelength measurement with 575 as an isosbestic point. Absorbance maxima for the two forms are at 561 and 566 nm, but the signals overlap considerably so that redox changes of the mixture are most readily obtained by measuring absorbance at 562 nm relative to 575 nm. The extinction coefficient value for 562 nm minus 575 nm is 23/mM/cm.

Download

Copyright Disclaimer:
This site does not store any files on its server. We only index and link to content provided by other sites. Please contact the content providers to delete copyright contents if any and email us, we'll remove relevant links or contents immediately.