Spectral analysis of the bc(1) complex components in situ: beyond the traditional difference approach

The cytochrome (cyt) bc(1) complex (ubiquinol: cytochrome c oxidoreductase) is the central enzyme of mitochondrial and bacterial electron-transport chains. It is rich in prosthetic groups, many of which have significant but overlapping absorption bands in the visible spectrum. The kinetics of the cytochrome components of the bc(1) complex are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. This difference-wavelength (DW) approach has been used extensively in the development and testing of the Q-cycle mechanism of the bc(1) complex in Rhodobacter sphaeroides chromatophores. However, the DW approach does not fully compensate for spectral interference from other components, which can significantly distort both amplitudes and kinetics. Mechanistic elaboration of cyt bc(1) turnover requires an approach that overcomes this limitation. Here, we compare the traditional DW approach to a least squares (LS) analysis of electron transport, based on newly determined difference spectra of all individual components of cyclic electron transport in chromatophores. Multiple sets of kinetic traces, measured at different wavelengths in the absence and presence of specific inhibitors, were analyzed by both LS and DW approaches. Comparison of the two methods showed that the DW approach did not adequately correct for the spectral overlap among the components, and was generally unreliable when amplitude changes for a component of interest were small. In particular, it was unable to correct for extraneous contributions to the amplitudes and kinetics of cyt b(L). From LS analysis of the chromophoric components (RC, c(tot), b(H) and b(L)), we show that while the Q-cycle model remains firmly grounded, quantitative reevaluation of rates, amplitudes, delays, etc., of individual components is necessary. We conclude that further exploration of mechanisms of the bc(1) complex, will require LS deconvolution for reliable measurement of the kinetics of individual components of the complex in situ.