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

Abstract

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.

Photocycle of a cyanobacteriochrome: a charge defect on ring C impairs conjugation in chromophore

“…Moreover, it has been known that a positively charged pyrrole nitrogen like N23 is conjugated with the π-system and that the formation of a carbocation at C12 would interrupt the bond length alternation of the conjugated system. 45,47 The latter statement is also borne out by the development of a greater degree of σ-bond character of the C–C bonds around C11 on the pathway towards Pg, as implied by the associated Δ δ 33 pattern of this carbon (Pr → Pg, Fig. 5D).…”

“…Such a conjugation defect localized around C12 would weaken the conjugation throughout the two inner rings, therefore leading to a blue shi of the absorption maximum. 21,47 Although the Lumi-R intermediate even displays the onset of a positive charge delocalization into ring C and also generation of a resonance-stabilized carbocation intermediate at C12, the ionic interaction offered by the C-ring propionate carboxylate seems to be fully functional until the nal transition to form Pg (Fig. 6).…”

Protein–chromophore interactions controlling photoisomerization in red/green cyanobacteriochromes

“…NpR6012g4 is a well-characterized protein with a stable photoproduct state [36,45,54,55,90,96,98,[107][108][109][110], but the equivalent domain (AnPixJg4) instead gives rise to a very unstable photoproduct and hence can function as a sensor of light intensity [46]. AnPixJg2 is also well-characterized [33,46,65,85,[111][112][113][114][115][116], whereas NpR6012g2 is unstable and is only amenable to basic characterization [45]. AnPixJg3 is inactive, whereas NpR6012g3 undergoes facile photoconversion [45,85].…”

Computational identification of key residues regulating fluorescence emission in a red/green cyanobacteriochrome

“…Cyanobacteriochromes (CBCRs) are a part of the superfamily of PHYs found only in cyanobacteria 10,14 . Unlike other PHYs, CBCRs require only GAF domains for bilin binding and absorb wider wavelength light covering the ultraviolet‐to‐red region 7,10 .…”

“…Slr1393 from cyanobacterium Synechocystis sp. PCC6803 consists of three consecutive GAF domains, out of which only the third GAF domain (Slr1393g3) binds to PCB and shows reversible red/green photoconversion 8,14,26–29 . In a recent study, multiple CBCR variants from Slr1393g3 capable of solely binding to BV were generated by molecular evolution, and some of them displayed phenomenal far‐red/red (Pfr/Pr) photoconversion 30 .…”