The kinetics of reactions around the cytochrome bf complex studied in an isolated system

Abstract

The kinetics of oxidation and reduction of P700, plastocyanin, cytochrome f and cytochrome b-563 were studied in a reconstituted system consisting of Photosystem I particles, cytochrome bf complex and plastocyanin, all derived from pea leaf chloroplasts. Decyl plastoquinol was the reductant of the bf complex. Turnovers of the system were initiated by laser flashes. The reaction between oxidised P700 and plastocyanin was non-homogeneous in that a second-order rate coefficient of c. 5×10(-7) M(-1) s(-1) applied to 80% of the P700(+) and c. 0.7×10(7) M(-1) s(-1) to the remainder. In the presence of bf complex, but without quinol, the electron transfer between cytochrome f and oxidised plastocyanin could be described by a second-order rate coefficient of c. 4×10(7) M(-1) s(-1) (forward), and c. 1.6×10(7) M(-1) s(-1) (reverse). The equilibrium coefficient was thus 2.5. Unexpectedly, there was little reduction of cytochrome f (+) or plastocyanin(+) by electrons from the Rieske centre. With added quinol, reduction of cytochrome b-563 occurred. Concomitantly, electrons appeared in the oxidised species. It was inferred that either the Rieske centre was not involved in the high-potential chain of electron transfer events, or that, only in the presence of quinol, electrons were quickly passed from the Rieske centre to cytochrome f (+). Additionally, the presence of quinol altered the equilibrium coefficient for the cyt f/PC interaction from 2.5 to c. 5. The reaction between quinol and the bf complex was describable by a second-order rate coefficient of about 3×10(6) M(-1) s(-1). The pattern of the redox reactions around the bf complex could be simulated in detail with a Q-cycle model as previously found for chloroplasts.

Inhibitor binding to isolated chloroplast cytochrome bf complex

“…Isolated cyt bf complexes, PS I particles and PC were prepared from pea leaves (Hope et al 1994a), and suspended in a buffer containing (mM) sorbitol 150, MgC12 I0, ascorbate 1, HEPES/KOH 10, anthraquinone sulphonate 0.02, Mega-9 15-25, pH 7.5. Cuvette concentrations were typically: PS I, 0.25-0.3/aM, PC 1-2 #M and cyt bf 0.6--0.9 #M in cyt Figure 1, particularly to show the necessity of changing K p~ for data where inhibitor was present.…”

“…39 f. Since quinol was absent, and negligible signal attributable to cyt b563 was observed, a simpler, threewavelength deconvolution of flash-induced absorbancy changes due to c y t f a t 554 nm, PC at 575 nm and P700 at 543 nm was made from responses obtained at these three wavelengths (cf. Rich et al 1987;Hope et al 1994a). The signals obtained were the average of 8-16 flashes at each wavelength, at usually 0.2 Hz.…”

“…Rich, personal communication) are inferred to act by binding at or near p-sites. Use of a system including isolated cyt bfcomplex (Rich et al 1987;Hope et al 1994aHope et al ,b, 1995 gives the opportunity to study the binding of these inhibitors in the absence of quinol or quinone, the latters being potentially able to compete with the inhibitors for binding sites (see Rich et al 1991;Kramer et al 1994, for DBMIB).…”

Plastid Regulation of Lhcb1 Transcription in the Chlorophyte Alga Dunaliella tertiolecta

“…The initial down-regulation of Lhcb1 expression may be resulted from an immediate decrease of DpH/ATP production caused by the uncouplers. On the longer time scale, however, the uncouplers facilitate PQH 2 oxidation (Rees et al, 1989;Hope et al, 1994), resulting in a more oxidized cytochrome b 6 f complex, which appears to up-regulate Lhcb1 transcription.…”

An Arabidopsis Mutant with High Cyclic Electron Flow around Photosystem I (hcef) Involving the NADPH Dehydrogenase Complex

“…The pmf also acts as a major regulator of photosynthesis, slowing electron transfer at the cytochrome b 6 f complex (Hope et al, 1994;Takizawa et al, 2007) and triggering photoprotective q E quenching of excitation energy (Crofts and Yerkes, 1994). The q E response is activated by acidification of the lumen via the conversion of violaxanthin to zeaxanthin by violaxanthin deepoxidase (Gilmore, 1997) and protonation of the PsbS protein (Li et al, 2000).…”