Electron Transfer Kinetics in Purified Reaction Centers from the Green Sulfur Bacterium Chlorobium tepidum Studied by Multiple-Flash Excitation

Abstract

Reaction center preparations from the green sulfur bacterium Chlorobium tepidum, which contain monoheme cytochrome c, were studied by flash-absorption spectroscopy in the near-UV, visible, and near-infrared regions. The decay kinetics of the photooxidized primary donor P840(+), together with the amount of photooxidized cytochrome c, were analyzed along a series of four flashes spaced by 1 ms: 95% of the P840(+) was reduced by cytochrome c with a t(1/2) of approximately 65 micros after the first flash, 80% with a t(1/2) of approximately 100 micros after the second flash, and 23% with a t(1/2) of approximately 100 micros after the third flash; after the fourth flash, almost no cytochrome c oxidation occurred. The observed rates, the establishment of redox equilibrium after each flash, and the total amount of photooxidizable cytochrome c are consistent with the presence of two equivalent cytochrome c molecules per photooxidizable P840. The data are well fitted assuming a standard free energy change DeltaG degrees of -53 meV for electron transfer from one cytochrome c to P840(+), DeltaG degrees being independent of the oxidation state of the other cytochrome c. These observations support a model with two monoheme cytochromes c which are symmetrically arranged around the reaction center core. From the ratio of menaquinone-7 to the bacteriochlorophyll pigment absorbing at 663 nm, it was estimated that our preparations contain 0.6-1.2 menaquinone-7 molecules per reaction center. However, no transient signal due to menaquinone could be observed between 360 and 450 nm in the time window from 10 ns to 4 micros. No recombination reaction between the primary partners P840(+) and A(0)(-) could be detected under normal conditions. Such a recombination was observed (t(1/2) approximately 19 ns) under highly reducing conditions or after accumulation of three electrons on the acceptor side during a series of flashes, showing that the secondary acceptors can stabilize three electrons. From our data, there is no evidence for involvement of menaquinone in charge separation in the reaction center of green sulfur bacteria.

Mutations in Both Sides of the Photosystem I Reaction Center Identify the Phylloquinone Observed by Electron Paramagnetic Resonance Spectroscopy

“…After signal accumulation over several laser shots at a fixed magnetic field, the field was shifted and signal acquisition was repeated in order to construct the EPR spectrum of the transient P 700 ϩ A 1 Ϫ state. Flash Absorption Spectroscopy-Flash-induced absorption changes on a nanosecond time scale were measured as described previously (34), except that a laser diode emitting at 820 nm (SDL-5411-G1 from Spectra Diode Laboratories) was used as measuring light source. Excitation flashes at 532 nm were provided by a neodymium/yttrium-aluminumgarnet laser from Quantel (pulse duration, 300 ps; pulse energy, ϳ200 J/cm 2 ; repetition rate, 1 Hz).…”

Fourier transform infrared spectroscopy of special pair bacteriochlorophylls in homodimeric reaction centers of heliobacteria and green sulfur bacteria

“…The upshift of 29 cm -1 from the 1984 cm -1 peak is indicative of a larger degree of charge localization on one side. The isolated RC complex includes two copies of the Cytc protein (Kusumoto et al 1999;Oh-oka et al 1995).…”

An overview on chlorophylls and quinones in the photosystem I-type reaction centers

“…However, the essential role of MQ as A 1 has been challenged, because preparations completely devoid of MQ but capable of electron transfer to the FeS centers were obtained (Oh-oka et al 1993;Frankenberg et al 1996;Hager-Braun et al 1997;Permentier et al 2000). Further, Kusumoto et al (1999) found no evidence that MQ acts as A 1 in their RC, although one MQ molecule remained per RC. Thus the question as to the existence of quinone and the function as an electron acceptor between A 0 and F x in the RC of green sulfur bacteria has not been settled.…”