Light- and pH-dependent structural changes in cyanobacteriochrome AnPixJg2.

Cyanobacteriochromes (CBCRs) are phytochrome-related photosensory proteins that play an essential role in regulating phototaxis, chromatic acclimation, and cell aggregation in cyanobacteria. Here, we apply solid-state NMR spectroscopy to the red/green GAF2 domain of the CBCR AnPixJ assembled in vitro with a uniformly 13C- and 15N-labeled bilin chromophore, tracking changes in electronic structure, geometry, and structural heterogeneity of the chromophore as well as intimate contacts between the chromophore and protein residues in the photocycle. Our data confirm that the bilin ring D is strongly twisted with respect to the B-C plane in both dark and photoproduct states. We also identify a greater structural heterogeneity of the bilin chromophore in the photoproduct than in the dark state. In addition, the binding pocket is more hydrated in the photoproduct. Observation of interfacial 1H contacts of the photoproduct chromophore, together with quantum mechanics/molecular mechanics (QM/MM)-based structural models for this photoproduct, clearly suggests the presence of a biprotonated (cationic) imidazolium side-chain for a conserved histidine residue (322) at a distance of ~2.7 Å, generalizing the recent theoretical findings that explicitly link the structural heterogeneity of the dark-state chromophore to the protonation of this specific residue. Moreover, we examine pH effects on this in vitro assembled holoprotein, showing a substantially altered electronic structure and protonation of the photoproduct chromophore even with a small pH drop from 7.8 to 7.2. Our studies provide further information regarding the light- and pH-induced changes of the chromophore and the rearrangements of the hydrogen-bonding and electrostatic interaction network around it. Possible correlations between structural heterogeneity of the chromophore, protonation of the histidine residue nearby, and hydration of the pocket in both photostates are discussed.

Hydrogen Bond between a Tyrosine Residue and the C-Ring Propionate Has a Direct Influence on Conformation and Absorption of the Bilin Cofactor in Red/Green Cyanobacteriochromes.

Cyanobacteriochromes (CBCRs) are photoreceptors of the phytochrome superfamily showing remarkable variability in the wavelengths of the first electronic transition-sometimes denoted as Q band-compared to canonical phytochromes. Both classes carry the same cofactor, a bilin, but the molecular basis for the wide variation of their absorption properties is still a matter of debate. The interaction between the cofactor and the surrounding protein moiety has been proposed as a possible tuning factor. Here, we address the impact of hydrogen-bonding interaction between the covalently bound tetrapyrrole cofactor (phycocyanobilin, PCB) and a conserved tyrosine residue (Y302) in the second GAF (cGMP-specific phosphodiesterase, adenylyl cyclases, and FhlA) domain of the red-/green-switching CBCR AnPixJ (AnPixJg2). In the wild type, AnPixJg2 shows absorption maxima of 648 and 543 nm for the dark-adapted (Pr) and photoproduct (Pg) states, respectively. The Y302F mutation leads to the occurrence of an additional absorption band at 687 nm, which is assigned to a new spectroscopically identified sub-state called PIII. Similar spectral changes result upon mutating the Y302F-homologue in another representative red-/green-switching CBCR, Slr1393g3. Molecular dynamics simulations on the dark-adapted state suggest that the removal of the hydrogen bond leads to an additional PCB sub-state differing in its A- and D-ring geometries. The origin of the Q band satellite in the dark-adapted state is discussed.