Re-examination of the properties and function of the b cytochromes of the thylakoid cytochrome bf complex

Abstract

The thermodynamic properties and function of the cytochrome bf complex in spinach thylakoids were re-examined. The two b-cytochromes of the complex were found to have distinct midpoint potentials, distinct dependencies of midpoint potential with pH, and distinct absorbance spectra. Cytochrome b h and b l were found to have peak absorbances at 563.2 and 565 nm, respectively, and midpoint potentials at pH 7 of −45 mV ( n = 1) and −150 mV ( n = 1), respectively. The titrations also included a third cytochrome species with an absorbance peak at 560 nm and a midpoint potential at pH 7 of +47 mV ( n = 1). All three species showed only small changes in midpoint potential with pH. At high potential ( E h > +500 mV), flash excitation which produced plastoquinol in the plastoquinone pool, led to the reduction of the cyt b component with peak absorbance at 563.2 nm (i.e., cyt b h). This reduction was accompanied by an electrogenic movement of electrons across the thylakoid membrane, as observed by an accompanying electrochromic signal. When MOA-stilbene was added to slow the re-oxidation of cyt b, the same actinic illumination produced a reduction of cyt b h which was about 45% larger, and an electrochromic shift which was about 24% smaller, than the respective changes in the absence of the inhibitor. By comparing the amplitude of the slow electrogenic phase with the extent of cyt b reduction in the presence of MOA-stilbene, the electron transfer to cyt b h was calculated to traverse 70–80% of the dielectrically weighted distance across the thylakoid membrane. From these results, it is concluded that cyt b h is located towards the stromal side of the membrane. From the predicted folding structures of the cyt b protein, we conclude that cyt b l must be located on the lumenal side of the complex. The results show that a rapid electron transfer occurs between the quinol oxidizing (Q o) site of the complex and cyt b h, and are consistent with an electrogenic step between the two cyt b hemes, as expected for the operation of a Q-cycle; they argue against previous suggestions that electron transfer between the two cytochrome hemes is kinetically prohibited.