Three major classes of flavin photosensors, LOV domains, BLUF proteins and cryptochromes regulate diverse biological activities in response to blue-light. Recent studies of structure, spectroscopy and chemical mechanism have provided unprecedented insight into how each family operates at the molecular level. In general, the photoexcitation of the flavin cofactor leads to changes in redox and protonation states that ultimately remodel protein conformation and molecular interactions. For LOV domains, issues remain regarding early photochemical events, but common themes in conformational propagation have emerged across a diverse family of proteins. For BLUF proteins, photoinduced electron transfer reactions critical to light conversion are defined, but the subsequent rearrangement of hydrogen bonding networks key for signaling remain highly controversial. For cryptochromes, the relevant photocycles are actively debated, but mechanistic and functional studies are converging. Despite these challenges, our current understanding has enabled the engineering of flavoprotein photosensors for control of signaling processes within cells.

Entrainment of circadian rhythms in higher organisms relies on light-sensing proteins that communicate to cellular oscillators composed of delayed transcriptional feedback loops. The principal photoreceptor of the fly circadian clock, Drosophila cryptochrome (dCRY), contains a C-terminal tail (CTT) helix that binds beside a FAD cofactor and is essential for light signaling. Light reduces the dCRY FAD to an anionic semiquinone (ASQ) radical and increases CTT proteolytic susceptibility but does not lead to CTT chemical modification. Additional changes in proteolytic sensitivity and small-angle X-ray scattering define a conformational response of the protein to light that centers at the CTT but also involves regions remote from the flavin center. Reduction of the flavin is kinetically coupled to CTT rearrangement. Chemical reduction to either the ASQ or the fully reduced hydroquinone state produces the same conformational response as does light. The oscillator protein Timeless (TIM) contains a sequence similar to the CTT; the corresponding peptide binds dCRY in light and protects the flavin from oxidation. However, TIM mutants therein still undergo dCRY-mediated degradation. Thus, photoreduction to the ASQ releases the dCRY CTT and promotes binding to at least one region of TIM. Flavin reduction by either light or cellular reductants may be a general mechanism of CRY activation.redox | photolyase | protein-protein interaction

Cryptochromes (CRYs) entrain the circadian clocks of plants and animals to light. Irradiation of the cryptochrome (dCRY) causes reduction of an oxidized flavin cofactor by a chain of conserved tryptophan (Trp) residues. However, it is unclear how redox chemistry within the Trp chain couples to dCRY-mediated signaling. Here, we show that substitutions of four key Trp residues to redox-active tyrosine and redox-inactive phenylalanine tune the light sensitivity of dCRY photoreduction, conformational activation, cellular stability, and targeted degradation of the clock protein timeless (TIM). An essential surface Trp gates electron flow into the flavin cofactor, but can be relocated for enhanced photoactivation. Differential effects of Trp-mediated flavin photoreduction on cellular turnover of TIM and dCRY indicate that these activities are separated in time and space. Overall, the dCRY Trp chain has evolutionary importance for light sensing, and its manipulation has implications for optogenetic applications of CRYs.

Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pK a . Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions.light sensing | flavoprotein | photochemistry | redox | molecular dynamics C ryptochromes (CRYs) are flavin-binding proteins that perform a variety of sensory and catalytic functions in all kingdoms of life (1, 2). CRYs are closely related to the DNA photolyases (PLs), which catalyze light-driven redox reactions to break apart pyrimidine dimers in UV-damaged DNA (1, 2). CRYs and PLs share a conserved photolyase homology region that consists of an α-helical domain, which binds flavin adenine dinucleotide (FAD) and an α/β Rossman-fold domain, which sometimes binds a pteridine or deazaflavin antenna cofactor. CRYs also contain C-terminal extensions of variable sizes that contribute to their specific functions. The range of activities found for CRYs and PLs require that their flavin cofactors assume a broad range of redox, protonation, and excited states (1, 2).In the fruit fly Drosophila melanogaster, a type I cryptochrome (dCRY) is the primary light receptor of the circadian clock (1, 3). In response to blue light, dCRY coordinates interactions between Timeless (TIM) and the E3-ubiquitin ligase Jetlag (JET) (4). JETmediated proteolysis of TIM destabilizes its partner Period (PER). PER serves as the principal repressor of circadian gene expression and its degradation phase-shifts the clock (3). dCRY also catalyzes light-induced self-degradation that involves another E3-ligase: Brwd3 or RAMSHACKLE (5)...

Despite ongoing controversy, several strategic frameworks for defining chemicals of concern (e.g., persistent, bioaccumulative, toxic [PBT]; persistent, mobile, toxic [PMT]; persistent organic pollutant [POP]) share persistence as a key criterion. Persistence should be considered over the entire chemical life cycle from production to disposal, including hazardous waste management. As a case study, we evaluate persistence criteria in hazardous waste regulations in Washington state, USA, illustrate impacts on reported waste, and propose refinements in these criteria. Although Washington state defines persistence based on half‐life (>1 y) and specific chemical groups that exceed summed concentration thresholds in waste (i.e., >0.01% halogenated organic compounds [HOCs] and >1.0% polycyclic aromatic hydrocarbons [PAHs]), persistence is typically addressed with HOC and PAH evaluation but seldom with half‐life estimation. Notably, persistence is considered (with no specific criteria) in corresponding federal regulations in the United States (Resource Conservation and Recovery Act). Consequently, businesses in Washington state report annual amounts of state hazardous waste (including persistent waste) separately from federal hazardous waste. Total state‐only waste, and total state and federal waste combined, nearly doubled (by weight) from 2008 to 2018. For the period 2016 to 2018, persistence criteria captured 17% of state‐only waste and 2% of total state and federal waste combined. Two recommendations are proposed to improve persistence criteria in hazardous waste regulations. First, Washington state should consider aligning its half‐life criterion with federal and European Union PBT definitions (e.g., 60–120 d) for consistency and provide specific methods for half‐life estimation. Second, the state should consider expanding its list of persistent chemical groups (e.g., siloxanes, organometallics) with protective concentration thresholds. Ultimately, to the extent possible, Washington state should strive toward harmonizing persistence in hazardous waste regulations with corresponding criteria in global PBT, PMT, and POP frameworks. Integr Environ Assess Manag 2021;17:455–464. © 2020 SETAC