Femtosecond photodynamics of the red/green cyanobacteriochrome NpR6012g4 from Nostoc punctiforme. 2. reverse dynamics

Phytochromes are red/far-red photosensory proteins that utilize photoisomerization of a linear tetrapyrrole (bilin) chromophore to photoconvert reversibly between red- and far-red-absorbing forms (P(r) and P(fr), respectively). Cyanobacteriochromes (CBCRs) are related photosensory proteins with more diverse spectral sensitivity. The mechanisms that underlie this spectral diversity have not yet been fully elucidated. One of the main CBCR subfamilies photoconverts between a red-absorbing 15Z ground state, like the familiar P(r) state of phytochromes, and a green-absorbing photoproduct ((15E)P(g)). We have previously used the red/green CBCR NpR6012g4 from the cyanobacterium Nostoc punctiforme to examine ultrafast photodynamics of the forward photoreaction. Here, we examine the reverse reaction. Using excitation-interleaved transient absorption spectroscopy with broadband detection and multicomponent global analysis, we observed multiphasic excited-state dynamics. Interleaved excitation allowed us to identify wavelength-dependent shifts in the ground-state bleach that equilibrated on a 200 ps time scale, indicating ground-state heterogeneity. Compared to the previously studied forward reaction, the reverse reaction has much faster excited-state decay time constants and significantly higher photoproduct yield. This work thus demonstrates striking differences between the forward and reverse reactions of NpR6012g4 and provides clear evidence of ground-state heterogeneity in the phytochrome superfamily.     

Identification of sesquiterpene synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. strain PCC 7120

Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene synthases identified in Nostoc sp. strain PCC 7120 (terpene synthase NS1) and Nostoc punctiforme PCC 73102 (terpene synthases NP1 and NP2). The second terpene synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-α-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-α-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene synthases. Coexpression of P450 genes with their adjacent located terpene synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.     

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

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole bilin-binding photoreceptors of cyanobacteria that exhibit high spectral diversity, gaining attention in optogenetics and bioimaging applications. Several engineering studies on CBCRs were attempted, especially for designing near-infrared (NIR) fluorescent proteins with longer fluorescence wavelengths. However, despite continuous efforts, a key component regulating fluorescence emission property in CBCRs is still poorly understood. As a model system, we focused on red/green CBCR Slr1393g3, from the unicellular cyanobacterium Synechocystis sp. PCC 6803 to engineer Pr to get far-red light-emitting property. Energy profiling and pairwise structural comparison of Slr1393g3 variants effectively reveal the mutations that are critical to the fluorescence changes. H497 seems to play a key role in stabilizing the chromophore environment, especially the α3 helix, while H495, T499, and Q502 are potential key residues determining fluorescence emission peak wavelength. We also found that mutations of α2 and α4 helical regions are closely related to the chromophore binding stability and likely affect fluorescence properties. Taken together, our computational analysis suggests that the fluorescence of Slr1393g3 is mainly controlled by the stabilization of the chromophore binding pocket. The predicted key residues potentially regulating the fluorescence emission property of a red/green CBCR will be advantageous for designing improved NIR fluorescent protein when combined with in vitro molecular evolution approaches.     

A novel photoactive GAF domain of cyanobacteriochrome AnPixJ that shows reversible green/red photoconversion

We report the discovery of a novel cyanobacteriochrome, the green/red photoreceptor AnPixJ (All1069), isolated from the heterocyst-forming cyanobacterium Anabaena (Nostoc) sp. PCC 7120. Cyanobacteriochromes are a recently emerging tetrapyrrole-based photoreceptor superfamily that are distantly related to the conventional red/far-red photoreceptor phytochromes (Phys). The chromophore-binding domains of AnPixJ produced in cyanobacterial and Escherichia coli cells both showed a reversible and full photoconversion between a green-absorbing form (λ_max = 543 nm) and a red-absorbing form (λ_max = 648 nm). Denaturation analysis revealed that the green-absorbing form and the red-absorbing form covalently ligated phycocyanobilin with E-configuration and Z-configuration at the C15C16 double bond, respectively. Time-resolved spectral analysis showed the formation of the first intermediate state peaking at 680 nm from the dark-stable red-absorbing form. This step resembles the first photoconversion step from the red-absorbing form to the red-shifted lumi-R intermediate state of the Phys. These results suggest that the Pr of AnPixJ is almost equivalent to that of the Phys and starts a primary photoreaction with Z-to-E isomerization in a mechanism similar to that in the Phys, but is finally photoconverted to the unique green-absorbing form.     

Isolation and characterization of thylakoid membranes from the filamentous cyanobacterium Nostoc punctiforme

Nostoc   punctiforme strain Pasteur Culture Collection (PCC) 73102, a sequenced filamentous cyanobacterium capable of nitrogen fixation, is used as a model organism for characterization of bioenergetic processes during nitrogen fixation in Nostoc . A protocol for isolating thylakoid membranes was developed to examine the biochemical and biophysical aspects of photosynthetic electron transfer. Thylakoids were isolated from filaments of N.   punctiforme by pneumatic pressure-drop lysis. The activity of photosynthetic enzymes in the isolated thylakoids was analysed by measuring oxygen evolution activity, fluorescence spectroscopy and electron paramagnetic resonance spectroscopy. Electron transfer was found functional in both PSII and PSI. Electron transfer measurements in PSII, using diphenylcarbazide as electron donor and 2,6-dichlorophenolindophenol as electron acceptor, showed that 80% of the PSII centres were active in water oxidation in the final membrane preparation. Analysis of the membrane protein complexes was made by 2D gel electrophoresis, and identification of representative proteins was made by mass spectrometry. The ATP synthase, several oligomers of PSI, PSII and the NAD(P)H dehydrogenase (NDH)-1L and NDH-1M complexes, were all found in the gels. Some differences were noted compared with previous results from Synechocystis sp. PCC 6803. Two oligomers of PSII were found, monomeric and dimeric forms, but no CP43-less complexes. Both dimeric and monomeric forms of Cyt b ₆/ f could be observed. In all, 28 different proteins were identified, of which 25 are transmembrane proteins or membrane associated ones.     

A soluble 3D LC/MS/MS proteome of the filamentous cyanobacterium Nostoc punctiforme

Nostoc punctiforme is an oxygenic photoautotrophic cyanobacterium with multiple developmental states, which can form nitrogen-fixing symbioses with a variety of terrestrial plants. 3D LC/MS/MS shotgun peptide sequencing was used to analyze the proteome when N. punctiforme is grown in continuous moderate light with ammonia as the nitrogen source. The soluble proteome includes 1575 proteins, 50% of which can be assigned to core metabolic and transport functions. Another 39% are assigned to proteins with no known function, a substantially higher fraction than in the Escherichia coli proteome. Many expressed proteins protect against oxidative and light stress. Seventy-one sensor histidine kinases, response regulators, and serine/threonine kinases, individually and as hybrid, multidomain proteins, were identified, reflecting a substantial capacity to sense and respond to environmental change. Proteins encoded by each of the five N. punctiforme plasmids were identified, as were 10 transposases, reflecting the plasticity of the N. punctiforme genome. This core proteome sets the stage for comparison with that of other developmental states.     

Isolation and Characterization of the Small Subunit of the Uptake Hydrogenase from the Cyanobacterium Nostoc punctiforme

In nitrogen-fixing cyanobacteria, hydrogen evolution is associated with hydrogenases and nitrogenase, making these enzymes interesting targets for genetic engineering aimed at increased hydrogen production. Nostoc punctiforme ATCC 29133 is a filamentous cyanobacterium that expresses the uptake hydrogenase HupSL in heterocysts under nitrogen-fixing conditions. Little is known about the structural and biophysical properties of HupSL. The small subunit, HupS, has been postulated to contain three iron-sulfur clusters, but the details regarding their nature have been unclear due to unusual cluster binding motifs in the amino acid sequence. We now report the cloning and heterologous expression of Nostoc punctiforme HupS as a fusion protein, f-HupS. We have characterized the anaerobically purified protein by UV-visible and EPR spectroscopies. Our results show that f-HupS contains three iron-sulfur clusters. UV-visible absorption of f-HupS has bands ∼340 and 420 nm, typical for iron-sulfur clusters. The EPR spectrum of the oxidized f-HupS shows a narrow g = 2.023 resonance, characteristic of a low-spin (S = ½) [3Fe-4S] cluster. The reduced f-HupS presents complex EPR spectra with overlapping resonances centered on g = 1.94, g = 1.91, and g = 1.88, typical of low-spin (S = ½) [4Fe-4S] clusters. Analysis of the spectroscopic data allowed us to distinguish between two species attributable to two distinct [4Fe-4S] clusters, in addition to the [3Fe-4S] cluster. This indicates that f-HupS binds [4Fe-4S] clusters despite the presence of unusual coordinating amino acids. Furthermore, our expression and purification of what seems to be an intact HupS protein allows future studies on the significance of ligand nature on redox properties of the iron-sulfur clusters of HupS.     

Synergistic effect of deoxyanthocyanins from symbiotic fern Azolla spp. on hrmA gene induction in the cyanobacterium Nostoc punctiforme

The hrmA gene of the N2-fixing cyanobacterium Nostoc punctiforme functions in repressing the formation of transitory motile filaments, termed hormogonia, by plant-associated vegetative filaments. Here, we report that anthocyanins can contribute to induction of hrmA expression. Aqueous extract from fronds of the fern Azolla pinnata, a host of symbiotic Nostoc spp., was found to be a potent inducer of hrmA-luxAB in N. punctiforme strain UCD 328. The hrmA-luxAB inducing activities of A. pinnata, as well as Azolla filiculoides, were positively correlated with levels of frond deoxyanthocyanins. Analyses of the deoxyanthocyanins in frond extracts revealed, in order of predominance, an acetylated glycoside derivative of luteolinidin (m/z 475) and of apigeninidin (m/z 459) and minor amounts of a second luteolinidin derivative. At up to 150 microM, a purified preparation of deoxyanthocyanins only weakly induced hrmA-luxAB on its own, but mixtures with hrmA-luxAB inducers (A. filiculoides extract or the flavonoid naringin) synergistically doubled to tripled their inducing activities. These results suggest that appropriately localized deoxyanthocyanins could function in plant-mediated mechanisms for repressing Nostoc spp. hormogonium formation.     

Photoconversion mechanism of a green/red photosensory cyanobacteriochrome AnPixJ: time-resolved optical spectroscopy and FTIR analysis of the AnPixJ-GAF2 domain

The photoconversion mechanism of a green/red sensory cyanobacteriochrome AnPixJ was studied. The phycocyanobilin-binding second GAF domain of AnPixJ of Anabaena sp. PCC 7120 was expressed in Escherichia coli cells. The His-tagged AnPixJ-GAF2 domain exhibited photoconversion between the green- and red-absorbing forms, APg(543) and APr(648), respectively. We detected four intermediate states in the photocycle between them, as follows: APr(648) → red light → APr(648)* → (with a rise time constant τ(r) of <100 ns) R1(650-80) (with a decay time constant τ(d) of <1 μs) → R2(610) (τ(d) = 920 μs) → APg(543) → green light → APg(543)* → (τ(r) < 50 ns) G1(570) (τ(d) = 190 μs) → G2(630) (τ(d) = 1.01 ms) → APr(648). These intermediates were named for their absorption peak wavelengths, which were estimated on the basis of the time-resolved difference spectra and global analysis of the time courses. The absorption spectrum of APr(648) resembles that of the Pr form of the phytochrome, while all the other states showed peaks at 530-650 nm and had wider bandwidths with smaller peak amplitudes. The fastest decay phases of fluorescence from APr(648)* and APg(543)* gave lifetimes of 200 and 42 ps, respectively, suggesting fast primary reactions. The APg(543)-minus-APr(648) difference FTIR spectrum in an H(2)O medium was significantly different from those reported for the Pfr/Pr difference spectra in phytochromes. Most of the peaks in the difference spectrum were shifted in the D(2)O medium, suggesting the high accessibility to the aqueous phase. The interactions of the phycocyanobilin chromophore with the surrounding amino acid residues, which are fairly different from those in the GAF domain of phytochromes, realize the unique green/red photocycle of AnPixJ.     

Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

Background  

The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively.     

Cloning of two carotenoid ketolase genes from Nostoc punctiforme for the heterologous production of canthaxanthin and astaxanthin

For the heterologous synthesis of keto-carotenoids such as astaxanthin, two carotenoid ketolase genes crtW38 and crtW148, were cloned from the cyanobacterium, Nostoc punctiforme PCC 73102 and functionally characterized. Upon expression in Escherichia coli, both genes mediated the conversion of beta-carotene to canthaxanthin. However in a zeaxanthin-producing E. coli, only the gene product of crtW148 introduced 4-keto groups into the 3,3'-dihydroxy carotenoid zeaxanthin yielding astaxanthin. The gene product of crtW38 was unable to catalyze this reaction. Both ketolases differ in their interaction with a hydroxylase in the biosynthetic pathway from beta-carotene to astaxanthin.     

Structural diffusion properties of two atypical Dps from the cyanobacterium Nostoc punctiforme disclose interactions with ferredoxins and DNA

Ferritin-like proteins, Dps (DNA-binding protein from starved cells), store iron and play a key role in the iron homeostasis in bacteria, yet their iron releasing machinery remains largely unexplored. The electron donor proteins that may interact with Dps and promote the mobilization of the stored iron have hitherto not been identified. Here, we investigate the binding capacity of the two atypical Dps proteins NpDps4 and NpDps5 from Nostoc punctiforme to isolated ferredoxins. We report NpDps-ferredoxin interactions by fluorescence correlation spectroscopy (FCS) and fluorescence resonance energy transfer (FRET) methods. Dynamic light scattering, size exclusion chromatography and native gel electrophoresis results show that NpDps4 forms a dodecamer at both pH 6.0 and pH 8.0, while NpDps5 forms a dodecamer only at pH 6.0. In addition, FCS data clearly reveal that the non-canonical NpDps5 interacts with DNA at pH 6.0. Our spectroscopic analysis shows that [FeS] centers of the three recombinantly expressed and isolated ferredoxins are properly incorporated and are consistent with their respective native states. The results support our hypothesis that ferredoxins could be involved in cellular iron homeostasis by interacting with Dps and assisting the release of stored iron.     

A second conserved GAF domain cysteine is required for the blue/green photoreversibility of cyanobacteriochrome Tlr0924 from Thermosynechococcus elongatus

Phytochromes are widely occurring red/far-red photoreceptors that utilize a linear tetrapyrrole (bilin) chromophore covalently bound within a knotted PAS-GAF domain pair. Cyanobacteria also contain more distant relatives of phytochromes that lack this knot, such as the phytochrome-related cyanobacteriochromes implicated to function as blue/green switchable photoreceptors. In this study, we characterize the cyanobacteriochrome Tlr0924 from the thermophilic cyanobacterium Thermosynechococcus elongatus. Full-length Tlr0924 exhibits blue/green photoconversion across a broad range of temperatures, including physiologically relevant temperatures for this organism. Spectroscopic characterization of Tlr0924 demonstrates that its green-absorbing state is in equilibrium with a labile, spectrally distinct blue-absorbing species. The photochemically generated blue-absorbing state is in equilibrium with another species absorbing at longer wavelengths, giving a total of 4 states. Cys499 is essential for this behavior, because mutagenesis of this residue results in red-absorbing mutant biliproteins. Characterization of the C 499D mutant protein by absorbance and CD spectroscopy supports the conclusion that its bilin chromophore adopts a similar conformation to the red-light-absorbing P r form of phytochrome. We propose a model photocycle in which Z/ E photoisomerization of the 15/16 bond modulates formation of a reversible thioether linkage between Cys499 and C10 of the chromophore, providing the basis for the blue/green switching of cyanobacteriochromes.     

Functional characterization of the twin ZIP/SLC39 metal transporters, NpunF3111 and NpunF2202 in Nostoc punctiforme

The ZIP family of metal transporters is involved in the transport of Zn²⁺ and other metal cations from the extracellular environment and/or organelles into the cytoplasm of prokaryotes, eukaryotes and archaeotes. In the present study, we identified twin ZIP transporters, Zip11 (Npun_F3111) and Zip63 (Npun_F2202) encoded within the genome of the filamentous cyanobacterium, Nostoc punctiforme PCC73120. Sequence-based analyses and structural predictions confirmed that these cyanobacterial transporters belong to the SLC39 subfamily of metal transporters. Quantitative real-time (QRT)-PCR analyses suggested that the enzymes encoded by zip11 and zip63 have a broad allocrite range that includes zinc as well as cadmium, cobalt, copper, manganese and nickel. Inactivation of either zip11 or zip63 via insertional mutagenesis in N. punctiforme resulted in reduced expression of both genes, highlighting a possible co-regulation mechanism. Uptake experiments using ⁶⁵Zn demonstrated that both zip mutants had diminished zinc uptake capacity, with the deletion of zip11 resulting in the greatest overall reduction in ⁶⁵Zn uptake. Over-expression of Zip11 and Zip63 in an E. coli mutant strain (ZupT736::kan) restored divalent metal cation uptake, providing further evidence that these transporters are involved in Zn uptake in N. punctiforme. Our findings show the functional role of these twin metal uptake transporters in N. punctiforme, which are independently expressed in the presence of an array of metals. Both Zip11 and Zip63 are required for the maintenance of homeostatic levels of intracellular zinc N. punctiforme, although Zip11 appears to be the primary zinc transporter in this cyanobacterium, both ZIP’s may be part of a larger metal uptake system with shared regulatory elements.     

Myxol and 4-ketomyxol 2'-fucosides, not rhamnosides, from Anabaena sp. PCC 7120 and Nostoc punctiforme PCC 73102, and proposal for the biosynthetic pathway of carotenoids

We identified the molecular structures of carotenoids in some Anabaena and Nostoc species. The myxoxanthophyll and ketomyxoxanthophyll in Anabaena (also known as Nostoc) sp. PCC 7120, Anabaena variabilis IAM M-3, Nostoc punctiforme PCC 73102 and Nostoc sp. HK-01 were (3R,2'S)-myxol 2'-fucoside and (3S,2'S)-4-ketomyxol 2'-fucoside, respectively. The glycoside moiety of the pigments was fucose, not rhamnose. The major carotenoids were beta-carotene and echinenone, and the minor ones were beta-cryptoxanthin, zeaxanthin, canthaxanthin and 3'-hydroxyechinenone. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence of the genome in Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, we proposed a biosynthetic pathway for the carotenoids and the corresponding genes and enzymes. Since only zeta-carotene desaturase (CrtQ) from Anabaena sp. PCC 7120 and beta-carotene ketolase (CrtW) from N. punctiforme PCC 73102 have been functionally identified, the other genes were searched by sequence homology only from the functionally confirmed genes. Finally, we investigated the phylogenetic relationships among some Anabaena and Nostoc species, including some newly isolated species.     

Comparison of photosynthesis and productivity of Gunnera tinctoria Molina (Mirbel) with and without the phycobiont Nostoc punctiforme L.

Abstract. Marked increases in growth and nitrogen content were found with Gunnera tinctoria Molina (Mirbel) plants infected (+ Nostoc ) with the cyanobacterium Nostoc punctiforme L., in comparison to uninfected (— Nostoc ) plants and this was attributed to N₂-fixation by the phycobiont. Whilst host and symbiont can be grown separately, preliminary data indicates that the host plant is reliant on the cyanobacterium to meet its nitrogen requirements because it has little capacity to assimilate nitrate. Although the maximum light-saturated rate of photosynthesis was higher in the + Nostoc plants, there was no reduction in photosynthetic efficiency under lightlimiting conditions, despite marked differences in plant nitrogen status. Differences in photosynthetic rate were implicated as the major reason for the differences in plant productivity. Stomatal conductance was insensitive to changes in plant nitrogen status and did not parallel the variation in photosynthetic rates. The ecological significance of the largely invariant stomatal response and the consequences of differences in water and nitrogen-use efficiencies between + and — Nostoc plants is discussed.     

Characterization of the Nitrate-Nitrite Transporter of the Major Facilitator Superfamily (the nrtP Gene Product) from the Cyanobacterium Nostoc punctiforme Strain ATCC 29133

The products of the NpR1527 and NpR1526 genes of the filamentous, diazotrophic, fresh-water cyanobacterium Nostoc punctiforme strain ATCC 29133 were identified as a nitrate transporter (NRT) and nitrate reductase (NR) respectively, by complementation of nitrate assimilation mutants of the cyanobacterium Synechococcus elongatus strain PCC 7942. While other fresh-water cyanobacteria, including S. elongatus, have an ATP-binding cassette (ABC)-type NRT, the NRT of N. punctiforme belongs to the major facilitator superfamily, being orthologous to the one found in marine cyanobacteria (NrtP). Unlike the ABC-type NRT, which transports both nitrate and nitrite with high affinity, Nostoc NrtP transported nitrate preferentially over nitrite. NrtP was distinct from ABC-type NRT also in its insensitivity to ammonium-promoted regulation at the post-translational level. The nitrate reductase of N. punctiforme was, on the other hand, inhibited upon addition of ammonium to medium, lending ammonium sensitivity to nitrate assimilation.     

Characterization of the nitrate-nitrite transporter of the major facilitator superfamily (the nrtP gene product) from the cyanobacterium Nostoc punctiforme strain ATCC 29133

The products of the NpR1527 and NpR1526 genes of the filamentous, diazotrophic, fresh-water cyanobacterium Nostoc punctiforme strain ATCC 29133 were identified as a nitrate transporter (NRT) and nitrate reductase (NR) respectively, by complementation of nitrate assimilation mutants of the cyanobacterium Synechococcus elongatus strain PCC 7942. While other fresh-water cyanobacteria, including S. elongatus, have an ATP-binding cassette (ABC)-type NRT, the NRT of N. punctiforme belongs to the major facilitator superfamily, being orthologous to the one found in marine cyanobacteria (NrtP). Unlike the ABC-type NRT, which transports both nitrate and nitrite with high affinity, Nostoc NrtP transported nitrate preferentially over nitrite. NrtP was distinct from ABC-type NRT also in its insensitivity to ammonium-promoted regulation at the post-translational level. The nitrate reductase of N. punctiforme was, on the other hand, inhibited upon addition of ammonium to medium, lending ammonium sensitivity to nitrate assimilation.     

1H, 15N,and 13C chemical shift assignments of cyanobacteriochrome NpF2164g3 in the photoproduct state

Cyanobacteriochrome (CBCR) photosensory proteins are phytochrome relatives using bilin chromophores for light sensing across the visible spectrum. Structural information is not available for two of the four known CBCR subfamilies. NpF2164g3 is a member of one such subfamily, exhibiting a violet/orange photocycle. We report backbone NMR chemical shift assignments for the light-activated orange-absorbing state of NpF2164g3 (BMRB no. 19150).