Studies on well-coupled Photosystem I-enriched subchloroplast vesicles — characteristics and reinterpretation of single-turnover cyclic electron transfer

The contributions of ferredoxin, P-700, plastocyanin and the cytochromes c-554, and b-563 to single-turnover electron transfer in Photosystem (PS) I-enriched subchloroplast vesicles were deconvoluted by fitting the literature-derived spectra of these components to the observed absorption data at a series of wavelengths, according to a linear least-squares method. The obtained corresponding residuals showed that the applied component spectra were satisfactory. The deconvoluted signals of cytochromes c-554 and b-563 differed in some cases significantly from the classical dual-wavelength signals recording at 554–545 nm and 563–575 (or −572) nm, due to interference from other electron-transferring components. KCN, DNP-INT (2-iodo-6-isopropyl-3-methyl-2′,4,4′-trinitrodiphenyl ether), DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzo-quinone) and antimycin A all inhibited electron transfer, although antimycin and DBMIB inhibited only after a few turnovers of the cytochrome bf complex. Fast flash-induced reduction of cytochrome b-563 exclusively reflected oxidant-induced reduction. Fast electron flow from cytochrome c-554 to plastocyanin and P-700 resulted in an apparent rereduction of cytochrome c-554 that was slower than the reduction of cytochrome b-563. Model simulations indicate that under highly oxidizing conditions for the Rieske FeS centre and reducing conditions for cytochrome b-563, the semiquinone at the Q_z site cannot only reduce cytochrome b-563, but can also oxidize cytochrome b-563 and reduce the Rieske FeS centre. The effect of 10 μM gramicidin D was evaluated in order to determine the contributions by electrochromic absorption changes around 518 nm. Gramicidin left electron transfer, monitored in the 550–600 nm range, unchanged. The gramicidin-sensitive (membrane potential-associated) signal at 518 nm differed from the signals recorded in the absence of gramicidin at 518 nm or 518–545 nm, due to spectral interference from electron-transferring components in the latter signals. KCN, DBMIB and antimycin A affected both the fast and slow components of the electrochromic signal, but did not proportionally affect the initial electron transfer from P-700 to ferredoxin (charge separation in PS I). Not only the slow (10–100 ms) component of the 518 nm absorption change, but also part of the fast (less than 1 ms) component appears to minitor electrogenic events in the cytochrome bf complex.