Kinetic and thermodynamic analysis of the light-induced processes in plant and cyanobacterial phytochromes.

The light-induced processes of the biological photoreceptor phytochrome (recombinant phyA of oat and recombinant CphA from the cyanobacterium Tolypothrix PCC7601) have been investigated in a time-resolved manner in the temperature range from 0 to 30°C. Both proteins were heterologously expressed and assembled in vitro with phycocyanobilin. The Pr state of plant phytochrome phyA is converted to the Pfr state after formation of four intermediates with an overall quantum yield of ~18%. The reversal reaction (Pfr-to-Pr) shows several intermediates, all of which, even the first detectable one, exhibit already all spectral features of the Pr state. The canonical phytochrome CphA from Tolypothrix showed a similar intermediate sequence as its plant ortholog. Whereas the kinetics for the forward reaction (Pr-to-Pfr) was nearly identical for both proteins, the reverse process (Pr formation) in the cyanobacterial phytochrome was slower by a factor of three. As found for the Pfr-to-Pr intermediates in the plant protein, also in CphA all detectable intermediates showed the spectral features of the Pr form. For both phytochromes, activation parameters for both the forward and the backward reaction pathways were determined.

Photophysical properties of structurally and electronically modified flavin derivatives determined by spectroscopy and theoretical calculations.

Four different riboflavin (RF) derivatives, two electronically modified compounds (1- and 5-deazariboflavin, 1DRF and 5DRF) and two sterically modified compounds (7,8-didemethyl- and 8-isopropylriboflavin, DMRF and iprRF), were subjected to a combination of time-resolved measurements (absorption and fluorescence) and high-level quantum chemical investigations. Both alkyl-modified flavins showed similar fluorescence properties as the parent compound, yet 5DRF had a larger quantum yield of fluorescence (PhiF = 0.52) than RF (PhiF = 0.27). Interestingly, 1DRF did not show fluorescence at all under these steady state conditions. The triplet quantum yield was different for the modified flavins such that no triplet formation was found for 1DRF, whereas the other compounds all formed triplet states (PhiTR for 5DRF of 0.64 and 0.50 and 0.23 for iprRF and DMRF, respectively). The triplet states of the two alkyl-modified flavins decayed with similar time constants as the parent compound, whereas a shorter lifetime was measured for 5DRF (tauTR = 15 micros, compared to tauTR = 29 micros for RF). In the calculations, the flavin derivatives were modeled as lumiflavins, that is, without the ribityl chain. We conclude that for aqueous solutions of DMRF, iprRF, and 5DRF intersystem crossing (ISC) takes place from the S1 1(pipi*) to the T2 3(pipi*) state by a vibronic spin-orbit coupling mechanism, a process common to most flavins, whereas ISC is slow in excited 1DRF due to the absence of a close-by triplet state.