Conservation and Diversity in the Primary Reverse Photodynamics of the Canonical Red/Green Cyanobacteriochrome Family.

In this report, we compare the femtosecond to nanosecond primary reverse photodynamics (15EPg → 15ZPr) of eight tetrapyrrole binding photoswitching cyanobacteriochromes in the canonical red/green family from the cyanobacterium Nostoc punctiforme. Three characteristic classes were identified on the basis of the diversity of excited-state and ground-state properties, including the lifetime, photocycle initiation quantum yield, photointermediate stability, spectra, and temporal properties. We observed a correlation between the excited-state lifetime and peak wavelength of the electronic absorption spectrum with higher-energy-absorbing representatives exhibiting both faster excited-state decay times and higher photoisomerization quantum yields. The latter was attributed to both an increased number of structural restraints and differences in H-bonding networks that facilitate photoisomerization. All three classes exhibited primary Lumi-Go intermediates, with class II and III representatives evolving to a secondary Meta-G photointermediate. Class II Meta-GR intermediates were orange absorbing, whereas class III Meta-G had structurally relaxed, red-absorbing chromophores that resemble their dark-adapted 15ZPr states. Differences in the reverse and forward reaction mechanisms are discussed within the context of structural constraints.

Conservation and diversity in the secondary forward photodynamics of red/green cyanobacteriochromes.

Cyanobacteriochromes (CBCRs) are photosensitive proteins that are distantly related to the phytochrome family of photoreceptors and, like phytochromes, exhibit photoactivity initiated by the excited-state photoisomerization of a covalently bound bilin chromophore. The canonical red/green photoswitching sub-family is the most studied class of CBCRs studied to date. Recently, a comparative study of the ultrafast (100 fs-10 ns) forward photodynamics of nine red/green photoswitching CBCR domains isolated from Nostoc punctiforme were reported (S. M. Gottlieb, P. W. Kim, C.-W. Chang, S. J. Hanke, R. J. Hayer, N. C. Rockwell, S. S. Martin, J. C. Lagarias and D. S. Larsen, Conservation and Diversity in the Primary Forward Photodynamics of Red/Green Cyanobacteriochromes, Biochemistry, 2015, 54, 1028-1042). We extend this study by characterizing the secondary (10 ns-1 ms) forward photodynamics of eight red/green photoswitching CBCRs from N. punctiforme with broadband time-resolved absorption spectroscopy. We demonstrate that the dynamics of these representative red/green CBCRs can be separated into two coexisting pathways involving a photoactive pathway that is successful in generating the terminal light-adapted 15EPg population and an unsuccessful pathway that stalls after generating a meta-stable Lumi-Of intermediate. The photoactive pathway evolves through a similar mechanism from excitation of the dark-adapted 15ZPr state to generate a far-red absorbing Lumi-Rf and then via a succession of blue-shifting photointermediates to ultimately generate the 15EPg state. This suggests a steady deviation from planarity of the bilin chromophore during the dynamics. While, the general mechanism for this evolution is conserved among these CBCBs, the timescales of these dynamics deviate significantly. Only half of the characterized CBCRs exhibit the unproductive pathways due to photoexcitation of dark-adapted 15ZPo sub-population that upon photoexcitation generates a meta-stable Lumi-Of intermediate, which eventually decays back to the 15ZPo subpopulation. 15ZPo is ascribed the horizontal Asp657 configuration that disrupts H-bonding with the chromophore in the dark-adapted state; its presence can be identified via enhanced absorption of high-energy tail of the electronic absorption spectrum.

Conservation and diversity in the primary forward photodynamics of red/green cyanobacteriochromes.

Phytochromes are red/far-red photosensory proteins that detect the ratio of red to far-red light. Crucial to light regulation of plant developmental biology, phytochromes are also found in fungi, bacteria, and eukaryotic algae. In addition to phytochromes, cyanobacteria also can contain distantly related cyanobacteriochromes (CBCRs) that, like phytochromes, utilize the photoisomerization of a linear tetrapyrrole (bilin) chromophore to convert between two photostates with distinct spectral properties. CBCRs exhibit a wide range of photostates spanning the visible and even near-ultraviolet spectrum. In both phytochromes and CBCRs, biosynthesis initially yields a holoprotein with bilin in the 15Z configuration, and the 15E photoproduct can often revert to the 15Z photostate in the absence of light (dark reversion). One CBCR subfamily, red/green CBCRs, typically exhibits red-absorbing dark states and green-absorbing photoproducts. Dark reversion is extremely variable in red/green CBCRs with known examples ranging from seconds to days. One red/green CBCR, NpR6012g4 from Nostoc punctiforme, is also known to exhibit forward photoconversion that has an unusually high quantum yield at ∼40% compared to 10-20% for phytochromes and CBCRs from other subfamilies. In the current study, we use time-resolved pump-probe absorption spectroscopy with broadband detection and multicomponent global analysis to characterize forward photoconversion of seven additional red/green CBCRs from N. punctiforme on an ultrafast time scale. Our results reveal that red/green CBCRs exhibit a conserved pathway for primary forward photoconversion but that considerable diversity exists in their excited-state lifetimes, photochemical quantum yields, and primary photoproduct stabilities.