Delmar S. Larsen

We demonstrate that the Halorhodospira halophila (Hhal) photoactive yellow protein (PYP) is not representative of the greater PYP family. The photodynamics of the PYP isolated from Salinibacter ruber (Srub) is characterized with a comprehensive range of spectroscopic techniques including ultrafast transient absorption, photostationary light titrations, Fourier transform infrared, and cryokinetics spectroscopies. We demonstrate that the dark-adapted pG state consists of two subpopulations differing in the protonation state of the chromophore and that both are photoactive, with the protonated species undergoing excited-state proton transfer. However, the primary I₀ photoproduct observed in the Hhal PYP photocycle is absent in the Srub PYP photodynamics, which indicates that this intermediate, while important in Hhal photodynamics, is not a critical intermediate in initiating all PYP photocycles. The excited-state lifetime of Srub PYP is the longest of any PYP resolved to date (∼30 ps), which we ascribe to the more constrained chromophore binding pocket of Srub PYP and the absence of the critical Arg52 residue found in Hhal PYP. The final stage of the Srub PYP photocycle involves the slowest known thermal dark reversion of a PYP (∼40 min vs 350 ms in Hhal PYP). This property allowed the characterization of a pH-dependent equilibrium between the light-adapted pB state with a protonated cis chromophore and a newly resolved pG′ intermediate with a deprotonated cis chromophore and pG-like protein conformation. This result demonstates that protein conformational changes and chromophore deprotonation precede chromophore reisomerization during the thermal recovery of the PYP photocycle.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that exhibit photochromism between two states: a thermally stable dark-adapted state and a metastable light-adapted state with bound linear tetrapyrrole (bilin) chromophores possessing 15Z and 15E configurations, respectively. The photodynamics of canonical red/green CBCRs have been extensively studied; however, the time scales of their excited-state lifetimes and subsequent ground-state evolution rates widely differ and, at present, remain difficult to predict. Here, we compare the photodynamics of two closely related red/green CBCRs that have substantial sequence identity (∼68%) and similar chromophore environments: AnPixJg2 from Anabaena sp. PCC 7120 and NpR6012g4 from Nostoc punctiforme. Using broadband transient absorption spectroscopy on the primary (125 fs to 7 ns) and secondary (7 ns to 10 ms) time scales together with global analysis modeling, our studies revealed that AnPixJg2 and NpR6012g4 have comparable quantum yields for initiating the forward (^15ZP_r → ^15EP_g) and reverse (^15EP_g → ^15ZP_r) reactions, which proceed through monotonic and nonmonotonic mechanisms, respectively. In addition to small discrepancies in the kinetics, the secondary reverse dynamics resolved unique features for each domain: intermediate shunts in NpR6012g4 and a Meta-G_f intermediate red-shifted from the ^15ZP_r photoproduct in AnPixJg2. Overall, this study supports the conclusion that sequence similarity is a useful criterion for predicting pathways of the light-induced evolution and quantum yield of generating primary intermediate Φ_p within subfamilies of CBCRs, but more studies are still needed to develop a comprehensive molecular level understanding of these processes.

While cheminformatics skills necessary for dealing with an ever-increasing amount of chemical information are considered important for students pursuing STEM careers in the age of big data, many schools do not offer a cheminformatics course or alternative training opportunities. This paper presents the Cheminformatics Online Chemistry Course (OLCC), which is organized and run by the Committee on Computers in Chemical Education (CCCE) of the American Chemical Society (ACS)’s Division of Chemical Education (CHED). The Cheminformatics OLCC is a highly collaborative teaching project involving instructors at multiple schools who teamed up with external chemical information experts recruited across sectors, including government and industry. From 2015 to 2019, three Cheminformatics OLCCs were offered. In each program, the instructors at participating schools would meet face-to-face with the students of a class, while external content experts engaged through online discussions across campuses with both the instructors and students. All the material created in the course has been made available at the open education repositories of LibreTexts and CCCE Web sites for other institutions to adapt to their future needs.

In this report, we compare the femtosecond to nanosecond primary reverse photodynamics (^15EP_g → ^15ZP_r) 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-G_o intermediates, with class II and III representatives evolving to a secondary Meta-G photointermediate. Class II Meta-G_R intermediates were orange absorbing, whereas class III Meta-G had structurally relaxed, red-absorbing chromophores that resemble their dark-adapted ^15ZP_r states. Differences in the reverse and forward reaction mechanisms are discussed within the context of structural constraints.

The photocycle of a reversible photoisomerizing rhodopsin mimic (M2) is investigated. This system, based on the cellular retinoic acid binding protein, is structurally different from natural rhodopsin systems; but exhibits a similar isomerization upon light irradiation. More specifically, M2 displays a 15-cis to all-trans conversion of retinal protonated Schiff base (rPSB) and all-trans to 15-cis isomerization of unprotonated Schiff base (rUSB). Here we use hybrid Quantum Mechan-ics/Molecular Mechanics (QM/MM) tools coupled with time resolved and cryokinetic UV-Vis spectroscopy to investigate these isomerization processes. The results suggest that primary rPSB photoisomerization of M2 occurs around the C13=C14 double bond within 2 ps following an aborted-bicycle pedal (ABP) isomerization mechanism similar to natural microbial rhodopsins. The rUSB isomerization is much slower and occurs within 30 ps around the C15=N double bond. Our findings reveal the possibility to engineer naturally occurring mechanistic features into artificial rhodopsins and also constitute a step towards understanding the photoisomerization of UV pigments. We conclude by reinforcing the idea that the presence of the retinal chromophore inside a tight protein cavity is not mandatory to exhibit ABP mechanism.

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 ^15EP_g population and an unsuccessful pathway that stalls after generating a meta-stable Lumi-O_f intermediate. The photoactive pathway evolves through a similar mechanism from excitation of the dark-adapted ^15ZP_r state to generate a far-red absorbing Lumi-R_f and then via a succession of blue-shifting photointermediates to ultimately generate the ^15EP_g 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 ^15ZP_o sub-population that upon photoexcitation generates a meta-stable Lumi-O_f intermediate, which eventually decays back to the ^15ZP_o subpopulation. ^15ZP_o 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.

The reverse photodynamics analysis of the NpR3784 cyanobacteriochrome, a representative of the noncanonical red/green (R/G) CBCR 3784 family was studied by broadband transient absorption pump-probe spectroscopy. Both primary (100 fs to 10 ns) and secondary (10 ns to 1 ms) photodynamics was characterized over nine decades of time and complemented with temperature-jump cryokinetics measurements. In contrast to canonical R/G CBCRs, the NpR3784 reverse reaction appears heterogeneous yielding two spectrally distinct primary photoproducts, Lumi-Go and Lumi-Gr, which decay on different timescales. The two primary photoproducts of NpR3784 equilibrate on ̴ 40 ns timescale and subsequently propagate as a single intermediate population into Pr. Such heterogeneity could arise from differences in the direction of D-ring rotation, in chromophore protonation or hydrogen bonding, in the mobility of protein residues and solvent water nearby the chromophore, or some combination therein. We conclude that the structural heterogeneity responsible for the atypical photodynamics of NpR3784 differs from that present in the canonical R/G CBCR family.

Cyanobacteriochromes (CBCRs) are a diverse family of cyanobacterial photoreceptors distantly related to the phytochrome photoreceptors of land plants. At least two lineages of CBCRs have reacquired red-absorbing dark states similar to the phytochrome Pr resting state but are coupled to green-absorbing light-adapted states rather than the familiar far-red-absorbing light-adapted state of phytochrome. One such lineage, the canonical red/green (R/G) CBCRs that includes AnPixJg2 and NpR6012g4, has been extensively characterized structurally and spectroscopically. We here examine the forward Pr photodynamics of NpR3784, a representative member of the second R/G CBCR subfamily. Using broadband transient-absorption pump-probe spectroscopy, we characterize both primary (100 fs - 10 ns) and secondary (10 ns - 1 ms) forward (Pr→ Pg) photodynamics and compare the results to temperature-jump cryokinetics measurements. Our studies show that the primary isomerization dynamics occurs on a ~10-ps timescale, yet remarkably, the red-shifted primary Lumi-Rf photoproduct found in all photoactive canonical R/G CBCRs examined to date is not observed in NpR3784 at room temperature. These results demonstrate that CBCR photocycles arising via convergent evolution nonetheless can exhibit diverse reaction pathways.

The optical control of spin state is of interest in the development of spintronic materials for data processing and storage technologies. Photomagnetic effects at the single-molecule level have recently been observed in the thin film state at 300 K in photochromic cobalt dioxolenes. Visible light excitation leads to ring-closure of a photochromic spirooxazine bound to a cobalt dioxolene, which leads to generation of a high magnetization state. Formation of the photomagnetic state occurs through a photoisomerization-induced spin-charge excited-state process and is dictated by the spirooxazine ligand dynamics. Here, we report a mechanistic investigation by ultrafast spectroscopy in the UV-vis region of the photochemical ring-closing process in the parent spirooxazine, azahomoadamantylphenanthroline spirooxazine, and the photomagnetic spirooxazine cobalt–dioxolene complex. The cobalt appears to stabilize a photomerocycanine transient intermediate, presumably the TCC isomer, formed along the ground-state potential energy surface (PES). Structural changes associated with the TCC isomer induces formation of the high-spin Co(II) form, suggesting that magnetization dymanics can occur along the excited-state PES, leading to ultrafast switching on the ps time scale. We demonstrate the full ring closure of the spiro-oxazine ligand is not required to switch magnetization states which can be induced with a higher yielding isomerization reaction. The ability of this system to undergo optically induced spin state switching on the ps time scale in the solid state makes it a promising canididate for resistive nonvolatile memory technologies.

Phytochromes proteins utilize ultrafast photoisomerization of a linear tetrapyrrole chromophore to detect the ratio of red to far-red light. Femtosecond photodynamics in the PAS-GAF-PHY photosensory core of the Cph1 phytochrome from Synechocystis sp. PCC6803 (Cph1Δ) were resolved with a dual-excitation-wavelength-interleaved pump-probe (DEWI) approach with two excitation wavelengths (600 nm and 660 nm) at three pH values (6.5, 8.0, and 9.0). Observed spectral and kinetic heterogeneity in the excited-state dynamics were described with a self-consistent model comprised of three spectrally distinct populations with different protonation states (Pr-I, Pr-II, and Pr-III), each composed of multiple kinetically distinct sub-populations. Apparent partitioning among these populations is dictated by pH, temperature, and excitation wavelength, Our studies provide insight into photocycle initiation dynamics at physiological temperatures, implicate the low-pH/low temperature Pr-I state as the photoactive state in vitro, and implicate an internal hydrogen-bonding network in regulating photochemical quantum yield.