Light-induced Proton Release of Phytochrome Is Coupled to the Transient Deprotonation of the Tetrapyrrole Chromophore

Abstract

The P r 3 P fr phototransformation of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens and the structures of the biliverdin chromophore in the parent states and the cryogenically trapped intermediate Meta-R C were investigated with resonance Raman spectroscopy and flash photolysis. Strong similarities with the resonance Raman spectra of plant phytochrome A indicate that in Agp1 the methine bridge isomerization state of the chromophore is ZZZasa in P r and ZZEssa in P fr , with all pyrrole nitrogens being protonated. Photoexcitation of P r is followed by (at least) three thermal relaxation components in the formation of P fr with time constants of 230 s and 3.1 and 260 ms. H 2 O/D 2 O exchange reveals kinetic isotope effects of 1.9, 2.6, and 1.3 for the respective transitions that are accompanied by changes of the amplitudes. The second and the third relaxation correspond to the formation and decay of Meta-R C , respectively. Resonance Raman measurements of Meta-R C indicate that the chromophore adopts a deprotonated ZZE configuration. Measurements with a pH indicator dye show that formation and decay of Meta-R C are associated with proton release and uptake, respectively. The stoichiometry of the proton release corresponds to one proton per photoconverted molecule. The coupling of transient chromophore deprotonation and proton release, which is likely to be an essential element in the P r 3 P fr photoconversion mechanism of phytochromes in general, may play a crucial role for the structural changes in the final step of the P fr formation that switch between the active and the inactive state of the photoreceptor.Phytochromes are photoreceptors that utilize light as a source of information for controlling numerous biological processes (1, 2). The chromophore, a methine-bridged tetrapyrrole ( Fig. 1), acts as a photoswitch between two stable, spectrally distinct forms, denoted as P r and P fr according to the red and far-red absorption maxima, respectively. The P r /P fr interconversion is initiated by the rapid Z/E photoisomerization of the C-D methine bridge (3), followed by chromophore relaxations that are coupled to structural changes of the apoprotein (4). These structural changes are the trigger for signal transduction. Resonance Raman (RR) 2 and IR spectroscopy have provided valuable insight into light-induced chromophore and protein structural changes (e.g. see Refs. 5-10), but molecular and mechanistic details are not yet known and no crystal structure of a phytochrome has been reported so far.While phytochromes were originally thought to be restricted to plants, the discovery of these chromoproteins in cyanobacteria (11) and other bacteria points to the prokaryotic origin of this family of photoreceptors. In contrast to plant phytochromes, typical bacterial phytochromes are light-regulated histidine kinases. Despite the quite different regulatory functions (12, 13), plant and bacterial phytochromes exhibit structural and mechanistic similarities. The phytochromobilin chromophore of plant phytoc...

Structure of the Biliverdin Cofactor in the Pfr State of Bathy and Prototypical Phytochromes

“…In all cases, protein variants lacking the histidine kinase module were used. Previous comparative studies of Agp1 revealed identical RR spectra for proteins including and lacking the kinase module (31,32).…”

Unusual Spectral Properties of Bacteriophytochrome Agp2 Result from a Deprotonation of the Chromophore in the Red-absorbing Form Pr

“…Negative signals below 725 nm at late times reflect the pump pulse-induced depletion of the Pr state; positive signals above 725 nm display the formation of Pfr on the time scale up to several seconds. The absence of an isosbestic point indicates the presence of more than two molecular species involved as expected for the Pr half-cycle in phytochromes (Agp1 (14), PhyA (32,33), and Cph1 (12,34)). …”

“…Vibrational spectroscopy, NMR studies, and pH titrations combined with UV/visible spectroscopy have shown that the chromophore is typically protonated in the Pr and Pfr states and that Pr to Pfr photoconversion is accompanied by a transient proton release (12)(13)(14). Free phytochrome chromophores are unprotonated in neutral solution (15), and protonation under acidic conditions occurs at the nitrogen atom of ring B or ring C; i.e.…”

Two ground state isoforms and a chromophore D -ring photoflip triggering extensive intramolecular changes in a canonical phytochrome

“…Phytochromes are, however, also now known in photosynthetic prokaryotes including cyanobacteria (3, 4), nonphotosynthetic bacteria (5), and fungi (6). Generally, the phytochrome apoprotein binds an open-chain tetrapyrrole as a chromophore (7,8) to form the red-light absorbing Pr ground state (λ max ≈ 658 nm in case of cyanobacterial phytchrome Cph1 from Synechocystis 6803). Red light absorption photoactivates the molecule to form the photoactivated far-red-light absorbing Pfr state (λ max ≈ 702 nm for Cph1) via a series of intermediates (8-10).…”

“…Generally, the phytochrome apoprotein binds an open-chain tetrapyrrole as a chromophore (7,8) to form the red-light absorbing Pr ground state (λ max ≈ 658 nm in case of cyanobacterial phytchrome Cph1 from Synechocystis 6803). Red light absorption photoactivates the molecule to form the photoactivated far-red-light absorbing Pfr state (λ max ≈ 702 nm for Cph1) via a series of intermediates (8)(9)(10). Photoactivation is thought to be initiated by a double bond isomerization of the chromophore (10,11).…”