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Carotenoids constitute a large class of isoprenoids synthesized by all photosynthetic organisms, some bacteria, fungi and arthro-pods1. Global vitamin A deficiency in children has sparked worldwide efforts to increase the levels of provitamin A carotenoids in food-crop staples2. This goal rests on furthering knowledge of how plants control and biosynthesize carotenoids that can be converted in humans to vitamin A. Metabolic engineering and breeding of plants rich in particular carotenoids will continue to be an impor-tant objective for addressing the challenges of providing food secu-rity in a changing climate. Carotenoid functions are central to plant growth and development. The plant carotenoid-biosynthetic path-way is mediated by enzymes encoded in the nucleus and localized to chloroplasts or other plastids1,2. The carotenoid-biosynthetic reac-tions begin with formation of the colorless 15-cis-phytoene, which undergoes desaturation and isomerization of double bonds to create carotenoids with yellow, red and orange colors. The pathway requires an electron-transfer chain and plastoquinones to channel electrons and protons produced during desaturation mediated by phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS). PDS produces 9,15,9′-tri-cis-ζ-carotene, which must be isomerized at the 15-15′–cis carbon-carbon double bond to form 9,9′-di-cis-ζ-carotene, the sub-strate for a second desaturase, ZDS (Fig. 1a). Although light can par-tially mediate this cis-trans carbon-carbon isomerization, Z-ISO3,4 is essential, especially in tissues receiving no light exposure, such as the endosperm tissue, which has been targeted for improvement of provitamin A carotenoids in efforts to alleviate global vitamin A defi-ciency2. Plants with insufficient Z-ISO also grow poorly under the stress of fluctuating temperature5. Because climatic variations alter the need for photosynthetic and nonphotosynthetic carotenoids, Z-ISO facilitates plant adaptation to environmental stress, a major factor affecting crop yield. Thus, Z-ISO is essential for maximizing plant fitness in response to environmental changes and for promot-ing accumulation of provitamin A carotenoids in edible tissues.

Mutants blocked in Z-ISO function accumulate 9,15,9′-tri-cis-ζ-carotene, the putative Z-ISO substrate3. When the gene encoding Z-ISO is introduced into Escherichia coli cells producing 9,15,9′-tri-cis-ζ-carotene, this carotenoid is isomerized into the putative Z-ISO product, 9,9′-di-cis-ζ-carotene3. These data suggest that Z-ISO is required for isomerization of the 15-cis bond in 9,15,9′-tri-cis-ζ-carotene but not the 15-cis bond in 15-cis-phytoene. In E. coli exper-iments, the isomerization activity associated with Z-ISO occurred in the presence of several upstream carotenoid-biosynthetic enzymes needed to produce the Z-ISO substrate. Thus, the possibility remains that Z-ISO is not an independently acting enzyme but that it instead alters one of the other enzymes present to gain a catalytic function of isomerization. Here we present data demonstrating that Z-ISO is a bona fide enzyme that catalyzes isomerization through a unique mechanism requiring a redox-regulated heme cofactor. This discovery raises new questions regarding the control of carotenogenesis in plants.

RESULTSExpression, isolation and activity assays of Z-ISOTo directly test whether Z-ISO is a bona fide enzyme, we developed an in vitro assay with isolated Z-ISO from Zea mays and artificial liposomes containing the Z-ISO substrate. First, we purified the substrate from E. coli3 and combined it with lipids to form arti-ficial liposomes. Next, we overexpressed and purified Z-ISO as a TEV protease–cleavable maltose-binding protein (MBP) fusion (MBP

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The as-purified enzyme (considered to be oxidized), as well as heat-denatured Z-ISO, was not functional. Therefore Z-ISO catalyzed isomerization only when we conducted the reaction under reduc-ing conditions but not oxidizing conditions. The liposomes used for the in vitro assay were also essential because reactions lacking liposomes also did not work (data not shown).

Predicting Z-ISO structure and localizationTo gain insight into the mechanism of isomerization, we sought to identify catalytic motifs or other characteristic domains in Z-ISO. Our previous BLAST6 analysis suggested that, although Z-ISO is highly conserved in plants, it shares sequence homology (~76% similarity) with only NnrU, an uncharacterized membrane protein associated with nitric oxide (NO) metabolism in noncarotenogenic bacteria that perform denitrification3. In addition, a chloroplast- targeting sequence was previously identified in Z-ISO, thus suggest-ing that Z-ISO is a plastid-localized protein3. We could not identify any other motifs to suggest a mechanism for isomerization. Therefore, we used bioinformatic approaches to generate hypothe-ses on the location and function of Z-ISO and tested them further.

MEMSAT3 (ref. 7) predicted seven transmembrane (TM) domains in maize Z-ISO (Fig. 1c), with TM2–TM7 showing homology to the corresponding TM domains in NnrU3. In contrast to a func-tional Arabidopsis transcript (ZISO1.1), a shorter Arabidopsis tran-script (ZISO1.2)3 encodes a nonfunctional protein with one less TM domain at the C terminus. The effect of the deletion suggests

that the C-terminal TM domain is important for the function (for example, activity or proper folding) of Z-ISO.

To test the prediction that Z-ISO is targeted to the chloro-plast, we fused the gene encoding GFP downstream of the gene encoding Z-ISO, including its transit peptide. We then transiently expressed the fusion construct in maize leaf protoplasts. GFP flu-orescence confirmed that Z-ISO colocalized in the chloroplasts together with chlorophyll (Fig. 1d). In vitro chloroplast protein import demonstrated that Z-ISO is a chloroplast integral membrane protein (Supplementary Fig. 2a), as predicted by the topology predictions. When taken together, our observations suggest that Z-ISO is localized in chloroplast membranes. We also found that Z-ISO exists in a high-molecular-weight protein complex of about 480 kDa (Supplementary Fig. 2b), as similarly noted for other carotenoid enzymes8.

Next, we applied homology-modeling tools to look for struc-tural homologies missed by the BLAST analysis. We expected that homology modeling would be limited by the under-representation of membrane-protein structures in the Protein Data Bank, owing to the inherent difficulties in crystallizing membrane proteins. Homology modeling of Z-ISO with the Meta Server9 program mod-eled the residues of Z-ISO onto an integral membrane protein, the diheme cytochrome b subunit of quinol-fumarate oxidoreductase10. The fold-recognition program LOOPP11 predicted that Z-ISO might contain nonheme iron (described below). These programs are based on unique algorithms, and therefore the templates chosen for

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Figure 1 | Z-ISO is an isomerase and integral membrane protein localized to chloroplasts. (a) steps in the carotenoid-biosynthetic pathway in which Z-Iso catalyzes the cis-trans isomerization of the central 15-15′ carbon-carbon double bond of 9,15,9′-tri-cis-ζ-carotene (tri-cis), the product of PDs, to form 9,9′-di-cis-ζ-carotene (di-cis), the substrate of ZDs. (b) Z-Iso in vitro enzymatic assay with substrate-containing liposomes and purified MBP

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modeling by the programs were different. However, neither NnrU nor Z-ISO had been annotated as metalloproteins.

Detection of iron in Z-ISOTo test the prediction that Z-ISO is a metalloenzyme, we used induc-tively coupled plasma optical emission spectrometry (ICP-OES) to measure the metal content (Online Methods). The result showed that iron, but not calcium, copper, nickel, magnesium, manganese, molybdenum or zinc, is present in the MBP

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Figure 5 | Testing mutation of putative heme ligands on enzyme activity, heme binding and UV-vis spectrum of Z-ISO. (a) Isomerization activity, tested by functional complementation in E. coli, with carotenoid products measured by HPlC. Means ± s.d. for 3 technical replicates are shown. (b,c) uv-vis spectra normalized for absorbance at 280 nm for extracted mutant fusion proteins, which were as purified (oxidized) (b) or dithionite reduced (c). Peak maxima are listed in Supplementary Table 3.

by a common histidine. We also examined the MCD spectrum of the dithionite-reduced Z-ISO. MCD showed a single heme spe-cies in the reduced Z-ISO coordinated by bis-histidine (Fig. 4b). Importantly, the amount of reduced histidine-histidine heme was equivalent to the combined concentration of the histidine-histidine and histidine-cysteine heme seen in oxidized Z-ISO.

If two heme centers exist, there should be two separate proximal histidines. To distinguish between these alternate hypotheses, it was necessary to use another approach to identify all histidines in Z-ISO that could serve as heme ligands. We next searched for the specific residues that might function as Z-ISO heme ligands. We aligned available Z-ISO sequences, identified all evolutionarily conserved residues that have been reported to serve as heme ligands28, muta-genized them to alanine and tested them for activity with E. coli complementation3. Of all conserved histidines, only two (H150 and H266) were required for activity (Fig. 5a and Supplementary Table 2). Mutants with alanine substitution at H150 (H150A) or H266 (H266A), as compared to wild-type Z-ISO, decreased the conversion of substrate to product. Loss of the isomerization activity was not due to absence of expression, the possibility of which we ruled out with an anti–maize Z-ISO polyclonal antiserum (Supplementary Fig. 8). Loss of either residue also disrupted heme binding, as evidenced by the decreased levels of bound heme for MBP-fusion proteins carrying the alanine variants and by the UV-vis spectral shift seen for both the as-purified (oxidized) or dithionite-reduced proteins (Fig. 5b,c).

On the basis of the mutagenesis results, we were able to rule out the two-heme model for Z-ISO. Two hemes would have necessitated a total of at least three required histidines (two proximal and at least one distal), but we found no additional conserved histidines that were required for activity beyond H150 and H266 (Supplementary Table 2). Predicted locations of H150 and H266 from the Z-ISO homology model (Fig. 6a) are consistent with coordination of a common cofactor. Therefore the data are consistent with the presence of a single heme that undergoes a change in axial ligation when reduced (Fig. 6b).

Does the heme ligand c263 function in isomerization?The ability of Z-ISO to bind exogenous ligands indicates the avail-ability of an axial coordination site on its heme and facile disso-ciation of one of two axial ligands. The most extensive dissociation takes place in the reduced, active form of the enzyme. Z-ISO activity is predicated on the heme ligands H150 and H266 and on the heme iron being in the reduced state. Therefore, it is possible that the heme iron directly mediates isomerization by interacting with the substrate. An alternative hypothesis is that, as a result of

redox-dependent ligand switching, the switch to bis-histidine exposes C263, which becomes accessible to mediate catalysis. A precedent for function of a cysteine residue in catalysis, particularly in double-bond isomerization, has been seen for isopentenyl diphosphate isomerase, a nonheme enzyme that cata-lyzes double-bond isomerization29. The C263 alternate heme ligand is the only cysteine in Z-ISO, and it is evolutionarily conserved in all Z-ISO sequences. The cysteine residue appears unlikely to function in protein dimerization because the in vitro reaction included the reducing agent dithiothreitol, which would eliminate dimerization mediated by cysteine sulfhydryl bridges. If C263 is essential for catalysis, then mutagenesis to a nonredox active residue should inactivate the enzyme. Mutation to alanine had no effect on activity or expression when we used the E. coli comple-

mentation system (Fig. 5a and Supplementary Fig. 8). Although the C263A MBP-fusion variant carries a smaller amount of heme that is the same as that in the H266A variant, the UV-vis spec-trum of C263A is similar to that of wild-type Z-ISO (Fig. 5b,c and Supplementary Table 3). When taken together, these results sug-gest that C263 is not catalytic but instead has a role in heme binding and reversible heme ligation.

The heme is likely to function as the mechanistic cofactor, according to the observations that with loss of either of the apparent histidine ligands (H150 or H266) Z-ISO becomes inactive, the heme spectrum is altered, and heme binding is decreased. EPR and MCD spectroscopy together identify the axial ligands as bis-histidine or histidine-cysteine. Notably, H266 and C263 are only three residues apart. Therefore, these two residues are probably the labile ligands that can exchange with each other in the ferric state, whereas H150 is the tightly associated proximal ligand that always remains bound to the heme regardless of different redox or binding events (Fig. 6a,b). EPR spectra show a small amount of histidine-ligated, pentacoordinate, high-spin heme, a possible intermediate during the ligand exchange. The presence of this high-spin species with a coordination vacancy, in equilibrium with the two different hexacoordinate ligation states of the low-spin heme, is consistent with the observation that CN− can bind to the ferric heme of Z-ISO. Furthermore, the substoichiometric binding of CN− is consistent with the MCD calibration data (Supplementary Fig. 7) showing that the pentacoordinate, high-spin species is likely to be less than 10–20% of the total heme. When Z-ISO is reduced to the Fe(ii) form, the heme ligand set becomes solely bis-histidine, thus sug-gesting a redox-dependent ligand switch. It is this reduced form that is active in vitro. The Fe(ii) heme can bind NO and CO, which we used as diagnostic probes to experimentally interrogate the heme for available coordination sites needed to coordinate an exogenous ligand (Fig. 6b).

DIScUSSIONWe have shown that Z-ISO is an integral membrane isomerase that responds to redox state in performing a key step in carotenoid biosynthesis. Isomerization is dependent on a unique cofactor carried by Z-ISO, a heme that undergoes redox-dependent ligand switching. We propose that reduction of the heme iron switches coordination of the heme to bis-histidine and exposes the active site for substrate binding. In the proposed mechanistic model (Fig. 6c), binding of the Z-ISO substrate displaces the weakly associated H266 ligand, and the π electrons of the 15-15′-cis carbon-carbon double bond in the substrate serve as a Lewis base for coordination with the ferrous heme iron of Z-ISO. There is a precedent for coordination

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between a carbon-carbon double-bond moiety and a heme iron, as reported for a bacterial flavohemoglobin30. Spectroscopic evidence provides support for coordinate bonding between the iron of the histidine-coordinated heme and a carbon-carbon double bond of an unsaturated lipid. Binding to a transition metal such as iron can reduce the bond order of an alkene because the π electrons are delocalized into an empty orbital on the metal31. As a result of direct coordination between the ferrous heme iron of Z-ISO and the target double bond in the substrate, the single σ bond remaining in the substrate would be free to rotate to the energetically more favorable trans configuration, thus converting 9,15,9′-cis-ζ-carotene to 9,9′-cis-ζ-carotene. As a consequence of cis-trans isomeriza-tion, the entire structure of the 40-carbon ζ-carotene substrate would change from a bulky W shape to a streamlined linear shape (Fig. 1a). These cis and trans geometrical isomers would interact uniquely with the microenvironment of the Z-ISO protein structure and contribute distinctly to membrane-lipid fluidity. Therefore, we predict that the altered carotenoid structure would drive release of the product from Z-ISO, thus allowing further enzymatic conversions of the Z-ISO product by downstream enzymes. Notably, according to the hard-soft acid-base theory32,

Fe(ii), in comparison to Fe(iii), is a soft Lewis acid and thereby prefers ligation to soft Lewis bases. Given that the Z-ISO substrate is a soft Lewis base, we anticipate that the ferrous state of Z-ISO presents superior binding kinetics and reactivity than the ferric state. In addition, coordination of the carotenoid double bond to the Fe(II) ion of the reduced Z-ISO heme forms a stable 18-electron coordination com-plex. In contrast, an unsaturated and less stable (17-electron) coordination complex would be generated if the substrate were coordinating electrons with the Fe(III) ion present in the as-purified Z-ISO heme. Thus, other than the aforementioned redox-dependent con-formational changes, the hard-soft acid-base analysis and the 18-electron rule in organome-tallic chemistry further explain the molecular basis for redox control of the isomerization activity of Z-ISO.

Heme-dependent carbon-carbon double-bond isomerization is rarely reported in the literature. The only other double-bond isomerase known to use heme as a cofactor is a bacterial cis-trans fatty-acid isomerase (CTI)33. CTI is a periplasmic enzyme that uses a c-type heme to perform a similar cis-trans isomerization of a double bond. However, little is known regarding the electronic structure or ligand-coordination state of the heme iron in this enzyme. The hypothesized catalytic mechanism of CTI is distinct from that of Z-ISO. It has been proposed that CTI func-tions in the oxidized ferric state and that the isomerization reaction is triggered by single-electron transfer from the double bond to the heme iron, thus oxidizing the double bond to a single bond33.

Our data show that reduction of the Z-ISO heme iron from Fe(iii) to Fe(ii) is necessary for enzyme activity. The heme reduction causes a ligand switch to bis-histidine and possibly trig-gers additional conformational changes at the active site of Z-ISO to allow substrate binding. In the resting ferric state, Z-ISO is postulated

to be in a closed conformation excluding the binding of the bulky substrate (Fig. 1a). Such redox-dependent ligand-switch phenom-ena have been observed in many other hemoproteins, and the purpose of the ligand-switch behavior is to induce conformational changes that drive functional activation. This strategy appears to be a common natural approach to control the functional activity of hemoproteins through redox changes. For example, cytochrome cd1 nitrite reductase must be reduced to become catalytically active through a mechanism that involves a redox-mediated heme-iron ligand switch34. Upon reduction, a tyrosine ligand of the d1 heme in that enzyme is displaced to generate a coordinate vacancy for substrate binding. Similarly, the CO gas–sensing transcription fac-tor CooA contains a heme cofactor that undergoes a ligand switch to make CooA competent for DNA binding20. Like Z-ISO, CooA goes through a redox-mediated ligand switch upon reduction of the heme iron: a cysteine axial ligand is replaced by a histidine, thus enabling the binding of CO to the heme iron at the ferrous state via displacement of the relatively weakly bound histidine ligand. Conformational changes then follow to drive DNA binding. Another example is bacterial di-heme cytochrome c peroxidase (bCcP)35. In the resting di-ferric state of bCcPs, one heme has a bis-histidine

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Figure 6 | Proposed mechanism of ligand rearrangement leading to active isomerization. (a) Z-Iso–homology model illustrating how proximity of alternate heme ligands predicts feasibility of distal-ligand switching between H266 and C263 when the heme proximal ligand is H150. (b) Proposed heme-ligand states based on experimental evidence. alternate ligand states for the Z-Iso heme (histidine-histidine or histidine-cysteine) were predicted from the MCD and EPR data. Diagnostic probes, No, Co and CN− were used to reveal specific states of the as-purified or dithionite-reduced enzyme. Cyanide (CN−) was used to capture the high-spin monoligated intermediate. Binding of No and Co (No/Co) to Z-Iso revealed available coordination sites on the heme originating from a weakly bound distal ligand. (c) Model for Z-Iso isomerization. the Fe(ii) in the Z-Iso heme coordinates its electrons with the delocalized π electrons (shaded) of the 15-15′-cis carbon-carbon double bond of 9,15,9′-ζ-carotene. the resulting carbon-carbon bond becomes like a single bond in character and therefore is able to rotate to the thermodynamically favorable trans orientation.np

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article NATURE cHEmIcAL bIOLOgy doi: 10.1038/nchembio.1840axial ligand set, and the other heme has a histidine-methionine axial ligand set. The two hemes are over 14 Å apart. A reductive activa-tion process is generally needed for the proper function of bCcPs: single-electron reduction of the high-potential histidine-methionine heme triggers a series of conformational changes that remotely dis-place one of the histidine ligands of the other heme, thus allowing the access of the cosubstrate, H2O2, to that site. Notably, a common feature of these examples is that reduction of the inactive ferric form generates the active ferrous form, and the ligand switch, as well as associated conformational changes, enables the binding of substrate via the creation of a coordinate vacancy, a weakly associated ligand or a binding cavity. This strategy can effectively protect the heme cofactor from nonproductive binding events and thereby avoids undesired side reactions.

If the activity of Z-ISO is controlled by redox state, then how might plastid physiology and stress affect Z-ISO and downstream flux through the carotenoid pathway? Plastids undergo dramatic shifts in redox status as a result of photosynthetic activity in the light and nonphotosynthetic activity in the dark. Changes in redox status are known to be reflected through dynamic control of metabolism. For example, redox modulators (such as ferrodoxins and thioredox-ins) adjust heme- and chlorophyll-biosynthetic activity in response to varying redox state36. It has been proposed that carotenoid biosyn-thesis is also under redox control, although most of the molecular details are unknown37. Mutations that inhibit expression of Z-ISO are already known to block production of carotenoid-pathway end products3,4. On the basis of the results presented here, we predict that changes in plastid redox state will directly influence Z-ISO activity and consequentially alter flux in the carotenoid-biosynthetic pathway. Redox tuning of Z-ISO activity could position Z-ISO as a gatekeeper for dynamic control of carotenogenesis on short time scales. That is, carotenoid pools could be rapidly adjusted by redox tuning of Z-ISO to respond to variable needs for photosynthesis and signaling pathways related to stress and development.

Stress is a known factor affecting biosynthesis and action of carotenoids and their derivatives38–41. NO is known to be produced directly at the site of carotenoid biosynthesis in plant plastids in response to stress42 and has been shown to inhibit carotenoid accumulation43. In addition, NO is known to inhibit heme enzymes through binding to the heme iron44, especially the ferrous form45. The ability of Z-ISO to bind NO, tested in the laboratory as a diagnostic heme-ligand probe, suggests that Z-ISO could be regulated by NO in vivo. Further study of Z-ISO will be critical for advancing the limited understanding of post-translational regulation of carotenogenesis.

Hemoproteins possess a wide range of biological functions, acting as enzymes, electron transporters, gas sensors, gas transporters and transcription factors, but double-bond isomerization is not generally considered to be a prototype activity for hemoproteins46. Z-ISO is the only known heme-dependent isomerase that uses a ferrous iron, undergoes redox-mediated ligand switching and performs isomerization of a long hydrocarbon in a membrane environment. Therefore, studies of Z-ISO as presented here open the path for further discovery and understanding of a new class of hemoenzymes that perform double-bond isomerization in hydro-phobic environments. In the case of Z-ISO, isomerization is critical for mediating metabolic flux of a vital plant pathway that is also important for nutrition of humans and other animals. Further understanding of Z-ISO function will provide opportunities to better control carotenoid biosynthesis and facilitate breeding of more-resilient plants in a changing climate and production of more-nutritious crops.

received 17 November 2014; accepted 13 May 2015; published online 15 June 2015

mETHODSMethods and any associated references are available in the online version of the paper.

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acknowledgmentsWe thank W. Hendrickson (Columbia University and New York Consortium on Membrane Protein Structure) for helpful discussions and use of the New York Structural Biology Center facilities. We thank M. Inouye (University of Medicine and Dentistry of New Jersey) for valuable advice on codon optimization and L. Bradbury (Lehman College and CUNY) for helpful discussions. E.T.W. was funded by the US National Institutes of Health (grant GM081160), CUNY and Lehman College. E.T.W. was also supported by travel funds from the Gordon Research Conferences to attend the Metals in Biology Gordon Research Conference, where E.T.W. was able to initiate collaborations with heme experts J.P. Hosler, A. Liu and J.H. Dawson through much-appreciated help from H. Gray (California Institute of Technology). The New York Consortium on Membrane protein structure (B.K. and J.D.L.) was supported through funds obtained from the US National Institutes of General Medical Sciences Protein Structure Initiative (PSI) program (grant GM095315). J.H.D. was funded by the US National Institutes of Health grant GM 26730; A.L. was funded by US National Institutes of Health grant R01GM108988 and the Georgia Research Alliance Distinguished Scholar Program. C.A.-D. was funded by The New Zealand Institute for Plant and Food Research Limited, New Zealand.

author contributionsWet-laboratory experiments were performed by J.B. (cloning, protein expression and purification, enzyme assays, HPLC, carotenoid analyses, mutagenesis, heme assays and UV-vis spectroscopy) and B.K. (cloning and protein expression), J.D.L. (design of pNYCOMPS vector, cloning, expression and protein-purification protocols) and J.P.H. (ICP-OES, UV-vis spectroscopy, heme assays and CO binding). EPR experiments were performed by J.G. and A.L. MCD experiments were performed by A.M., M. Sono and J.H.D. Localization and import, including related gene cloning, was conducted by M. Shumskaya. J.B. and E.T.W. performed bioinformatic analyses. C.A.-D. prepared mutant Z-ISO fusion proteins while on a short-term sabbatical in the Wurtzel laboratory. All authors contributed to data analysis and to the writing and editing of the manuscript.

competing financial interestsThe authors declare no competing financial interests.

additional informationSupplementary information is available in the online version of the paper. Reprints and permissions information is available online at http://www.nature.com/reprints/index.html. Correspondence and requests for materials should be addressed to E.T.W.

43. Chang, H.-L., Hsu, Y.-T., Kang, C.-Y. & Lee, T.-M. Nitric oxide down-regulation of carotenoid synthesis and PSII activity in relation to very high light-induced singlet oxygen production and oxidative stress in Chlamydomonas reinhardtii. Plant Cell Physiol. 54, 1296–1315 (2013).

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46. Munro, A., Girvan, H., McLean, K., Cheesman, M. & Leys, D. in Tetrapyrroles, Birth, Life and Death (eds. Warren, M. & Smith, A.) 160–183 (Landes Bioscience, Austin, Texas, USA, 2009).

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ONLINE mETHODSGeneral gene cloning. All gene constructs were verified by DNA sequencing.

Z-ISO expression and purification. Cloning. The maize Z-ISO coding sequence with transit sequence was commercially synthesized (Genscript) to be codon optimized for E. coli, and restriction sites were added for cloning into SacI and BamH1 sites of pUC57 (sequences in Supplementary Fig. 9). The final construct was named ZmZISO ACA-less (no. 516). From this clone, the sequence encoding Z-ISO beginning at residue 49 was PCR amplified with primers tacttccaatccaatgccatg CGTCCGGCGCGTGCGGTGG (forward) and TTATCCACTTCCAATG CTACCAGGGAAGTTGGTAGCTG (reverse) and inserted by ligation-independent cloning47 into pMCSG9-His10 (no. 646). Primer sequences in lowercase letters were for ligation-independent cloning, and those in uppercase were gene specific. The resulting construct, pMCSG9 Z-ISO E2 (no. 582), encodes a MBP

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(5′-atcggatccCCAGGGAAGTTGGTAGCTGGATGC), containing a BamHI site, and was inserted into the pUC35S-sGFP-Nos vector52 (digested with XbaI and BamHI), to produce the pUC35S-M-ZISO-sGFP-Nos plasmid (no. 568), which was used for transient expression. Transient expression of Z-ISO-GFP in maize green leaf protoplasts was performed as previously described52.

In vitro import of Z-ISO into chloroplasts. A full copy of the maize Z-ISO gene, without a stop codon, was amplified from pColZmZ-ISO1 (no. 497)3 with forward primer 2851 (ccacctgcaGAATTCtatggcctc), containing an EcoRI site, and reverse primer 2854 (gtcTCTAGAttatttttcaaattgaggatgagaccaccag-ggaagttggtagct), containing a streptavidin tag and XbaI site, and was inserted into vector pTnT (Promega), which was digested with the same restriction enzymes to yield plasmid pTnT-M-ZISO-Strep (no. 570). pTnT-M-ZISO-Strep was used as a template for in vitro protein synthesis. In vitro protein synthesis and import of Z-ISO into isolated pea chloroplasts were performed as previ-ously described52. After import, chloroplasts were treated with thermolysin (+) to remove nonspecifically bound protein. Chloroplasts were also fractionated into soluble (S) and membrane (M) fractions, including envelope and thyla-koid; an equal amount of the membrane fraction as in M was alkaline treated (MA) to remove peripheral membrane proteins, thus indicating that Z-ISO is a membrane integral protein. We previously showed that alkaline treatment will remove loosely associated peripheral membrane proteins rather than integral membrane proteins, which remain membrane associated52,53.

Identification of a Z-ISO complex. After [35S]methionine-labeled Z-ISO was imported into chloroplasts, the chloroplast sample was treated with 0.5% Triton X-100 to isolate protein complexes under native conditions. The sample was then separated into individual complexes by native gel electrophoresis in a NativePAGE Novex 4–16% gel (Invitrogen, Life Technologies), according to the instructions of the manufacturer. The gel was then dried and the radioactive band detected by a Phosphorimager system (Amersham, GE Life Sciences). The size of the band was estimated in comparison to NativeMark protein marker (Invitrogen, Life Technologies).

Detection of metals in Z-ISO. Inductively coupled plasma optical emission spectrometry (ICP-OES). Samples of MBP90% pure) were dialyzed three times each against a 1,000-fold volume of buffer (20 mM Tris, pH 7.6, 20 mM NaCl, 5% glycerol, 0.02% DDM, 0.1 mM DTT and 1 mM EDTA) and injected into a Spectro Genesis inductively coupled optical emission spectrom-eter to measure the concentrations of iron at 238.204 nm and 259.941 nm and sulfur at 180.731 nm, as previously described54. For 23 μM protein, 15.4 μM iron was detected. Levels of calcium, copper, nickel, magnesium, manganese, molybdenum or zinc were insignificant.

Detection of heme. Pyridine hemochrome assay. To determine whether the chromophore bound to Z-ISO was heme, a pyridine hemocrome assay was performed55. Purified protein (750 μl) was mixed with 75 μl of 1 N NaOH (Fisher Scientific), 175 μl of pyridine (Sigma-Aldrich) and 2 mg of sodium dithionite. The UV-vis absorption spectrum was immediately recorded and compared with the spectrum of the initial purified sample before addition of dithionite. The presence of the Soret band at 414 nm in the ferric state and the presence of the Soret band (418 nm) and appearance of the α-β bands at 555 and 530 nm, respectively, in the ferrous state were used as evidence for the presence of heme.

Heme stain. Heme staining, based on heme peroxidase activity, was per-formed essentially as previously reported56. Protein samples were separated on a NuPAGE Bis-Tris 12% polyacrylamide gel (Invitrogen). The gel was rinsed with water for 15 s and then incubated for 1 h in the dark in a solution contain-ing 30 ml of 40 mM TMBZ (3,3,5,5′-tetramethylbenzidine, Sigma-Aldrich) in methanol; this was followed by the addition of 70 ml of 0.25 M sodium acetate, pH 5.0 (Sigma-Aldrich). Then 5 ml of 3% hydrogen peroxide was added, and samples were mixed well until a signal corresponding to the MBP Z-ISO band appeared. The gel background was removed by destaining 15 min with 3:7 isopropanol/0.25 M sodium acetate.

Binding of CN−. MBP

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washes of 5 min each. Immunoreactions were visualized with the Super Signal West Dura kit (Thermo Scientific). Fluorescence signals were captured with a G:box (Chemi XT4) from Syngene with Genesys V1. 3.1.0 software.

10 mM IPTG and further incubation for 40 h at 28 °C with slow shaking (100 r.p.m.) and an additional 2 d without shaking. For carotenoid extraction, bac-terial cultures were centrifuged at 2,600g for 10 min. Pellets were resuspended in 5 ml of methanol containing 1% of butylated hydroxytoluene (Sigma-Aldrich, ≥99% pure) and sonicated with a Vibra Cell VC600 sonicator equipped with a 3-mm tapered microtip (Sonics & Materials) on ice twice (30 s each, at 60% power). Extracts were centrifuged at 2,600g for 10 min, supernatants were transferred to 15-ml conical tubes, and extracts were evaporated under nitro-gen gas in the dark. Dried samples were resolubilized in 500 μl of methanol, transferred to 1.5-ml tubes, frozen at −80 °C for 1 h and centrifuged at 16,000g at 4 °C, and supernatants were used for HPLC separation as described above. Complementation experiments were replicated three times.

Immunodetection of Z-ISO. For antibody generation, 2 mg of MBP

Supplementary Information

Control of carotenoid biosynthesis through a heme-based cis-trans isomerase Jesús Beltrán1,2, Brian Kloss3, Jonathan P. Hosler4, Jiafeng Geng5,9, Aimin Liu5, Anuja Modi6, John H. Dawson6, Masanori Sono6, Maria Shumskaya1, Charles Ampomah-Dwamena1,7, James D. Love3, 8, and Eleanore T. Wurtzel1,2*

1Department of Biological Sciences, Lehman College, The City University of New York, 250 Bedford Park Blvd. West, Bronx, NY 10468 2The Graduate School and University Center-CUNY, 365 Fifth Ave., New York, NY 10016-4309 3 New York Structural Biology Center, 89 Convent Avenue, New York, NY 10027-7556 4 Department of Biochemistry, University of Mississippi Medical Center, 2500 N. State St., Jackson, MS 39216-4500 5 Department of Chemistry, Georgia State University, 50 Decatur St. SE, Atlanta, Georgia 30303-2924 6 University of South Carolina, Department of Chemistry and Biochemistry, 631 Sumter Street, Columbia, SC 29208-0001 7 present address: The New Zealand Institute for Plant and Food Research Limited, Private Bag 92 169, Auckland 1142, New Zealand 8 present address: Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY, 10461 9 present address: School of Chemistry and Biochemistry, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, GA 30332-0363 *Corresponding author: Dr. Eleanore Wurtzel Department of Biological Sciences Lehman College, The City University of New York 250 Bedford Park Boulevard West Bronx, NY 10468 Tel.: 718-960-8643; Fax: 718-960-8236 E-mail: wurtzel@lehman.cuny.edu

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mailto:wurtzel@lehman.cuny.edu

Supplementary Results

Supplementary Tables Supplementary Table 1. Iron species in Z-ISO. Summary of the EPR parameters for the iron species present in as-purified Z-ISO. Iron species Ligands g factors High-spin heme His 5.8, 2.0 Low-spin heme #1 bis-His 2.98, 2.24a, 1.42 Low-spin heme #2 His-Cys 2.54, 2.24a, 1.86 Low-spin heme #3 His-Cys 2.50, 2.24a, 1.86 Low-spin heme #4 His-Cys 2.43, 2.24a, 1.92 Nonheme ironb N/D 4.3 aMultiple sets of overlapping signals bA minor species likely derived from non-specific iron binding N/D: not determined Supplementary Table 2. Mutagenesis of Z-ISO putative heme ligands. Conserved residues mutagenized to Alanine (A) and tested for activity by functional complementation in E. coli.

Mutagenesis of conserved

residue to alanine (A)

Whether required for Z-ISO function

H150A Yes H191A No H208A No H241A No H253A No

H354 A No H266A Yes H285A No H286A No C263A No

Supplementary Table 3. UV-Vis spectra of Z-ISO and mutants. Peak maxima (nm) from UV-Vis spectra of Z-ISO and variants. Data taken from Figure 5. oxidized reduced protein Z-ISO 414 425 530 559 H150A 406 427 525-528 560 H266A 406 426-427 519 560 C263A 413 425 530 559

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Supplementary Figures

Supplementary Figure 1. Z-ISO fusion protein. (a) Cartoon showing Z-ISO fusion protein containing histidine tag (His-Tag), maltose binding protein (MBP), TEV protease cleavage site, and Z-ISO. (b) Z-ISO fusion protein of 90% purity was analyzed by SDS-PAGE. MBP::Z-ISO (marked by arrow) was used for the in vitro assay (after cleavage) and for all subsequent spectroscopic analyses. Prestained molecular weight markers (kD) (SeeBlue® Plus2Pre-Stained Standard, Invitrogen) are shown in the left lane.

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Supplementary Figure 2. Z-ISO localization in chloroplasts. (a) In vitro import into pea chloroplasts. In vitro translated 38 kD Z-ISO is cleaved upon import to 33 kD, suggesting a 5 kD transit peptide. After import, chloroplasts were treated with thermolysin (+) to remove nonspecifically bound protein. Chloroplasts were also fractionated into soluble (S) and membrane (M) fractions; an equal amount of the membrane fraction as in M was alkaline-treated (MA) to remove peripheral membrane proteins. (b) Z-ISO is in a ~480 kD complex in chloroplasts as separated on a Blue Native gel.

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Supplementary Figure 3. Z-ISO contains heme iron. (a) Cell pellets expressing MBP::Z-ISO were brown colored compared with pellets expressing MBP only. (b) MBP::Z-ISO protein extracts were brown suggesting presence of a cofactor.

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Supplementary Figure 4. Heme staining of Z-ISO. Uncut gel showing heme and Coomassie staining of Z-ISO (see Fig. 2a). MBP::Z-ISO was treated with (+TEV) and without (-TEV) TEV protease.

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Supplementary Figure 5. NnrU pyridine hemochrome assay. NnrU (Agrobacterium tumefaciens C58) was expressed as a fusion protein in E. coli, purified, and treated with and without dithionite. A pyridine hemochrome assay of the extracts was analyzed by UV-visible absorption spectroscopy.

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Supplementary Figure 6. CN- binding to ferrous Z-ISO. MBP::Z-ISO, 75.46 KDa (1.58 mg/ ml, 21 µM) treated with dithionite, was incubated with KCN at a final concentration of 2 mM. UV visible spectra were recorded before and immediately after addition of KCN. The inset shows the difference spectrum.

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Supplementary Figure 7. MCD calibration of detection limits for high-spin heme. MCD and UV-Vis calibration spectra for low spin (ls, Cyt. b5)/high spin (hs, Mb) states of ferric [Fe(III)] heme proteins. From this figure, it is estimated that Z-ISO contains >80% low spin heme.

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Supplementary Figure 8. Verifying expression levels of Z-ISO and variants. Immunodetection of Z-ISO and Z-ISO variants (without transit peptide) expressed in E. coli cells harboring the pACCRT-EBP vector which confers accumulation of the Z-ISO substrate. The control sample is from cells containing the pACCRT-EBP vector alone. Upper panel: Western blot analysis using anti Z-ISO. Lower panel: Coomassie stain for protein samples used for immunodetection. One lane in both panels was blocked because the sample was unrelated to the paper.

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Maize Z-ISO, codon optimized for E coli Clone #516, named ZmZISO ACA-less DNA sequence Sac I/Bam H1 sites (in bold) added at 5’ and 3’ ends for cloning into pUC57). Z-ISO start codon is underlined. GAGCTCatggcgagccagctgcgtctgcatctggcggcgaccccgccgctgctgccgcatcgtcgtccgcatctgccgcgtccgctgtgcccgaccctgaacccgattcgtgcgccgctgccgccgctgagccgtgtgctgagccatgcgcgtccggcgcgtgcggtgggcggcggcattgaaccgaaagaaggcgtggtggcggaaggcgatgaaagcggcggcggcccggtgctggtgggcgaagatagcgcggcgtttgaactgaaagatcagagcgtggcgagctgggcgtattttgcgggcattctgggcgcggtgctggtggcgctgaacgtgctgtggattgatccgagcaccggcgtgggcaccaaatttctggatgcggtggcgagcgtgagcgatagccatgaagtggtgatgctgctgctgaccattatttttgcggtggtgcatagcggcatggcgagcctgcgtgaaagcggcgaaaaaattgtgggcgaacgtgtgtatcgtgtgctgtttgcgggcattagcctgccgctggcggtgaccaccattgtgtattttattaaccatcgttatgatggcacccagctgtggcaggtgcagggcattaccggcattcatgaactgctgtggtttagcagctttattagctttttttttctgtatccgagcacctttaacctgctggaagtggcggcggtggataaaccgaaactgcatatgtgggaaaccggcattatgcgtattacccgtcatccgcagatggtgggccaggtgatttggtgcctggcgcataccctgtggattggcaatagcgtggcggtggcggcgagcgtgggcctgattagccatcatctgtttggcgcgtggaacggcgatcgtcgtctgctgagccgttatggcgaagcgtttgaagtgctgaaaaaacgtaccagcgtgatgccgtttgcggcgattattgatggccgtcagaaactgccgaaagattatcataaagaattttttcgtctgccgtatgtggcgattaccatgctgaccctgggcgcgtattttgcgcatccgctgatgcaggcgagcagctatcagctgccgtggtaaGGATCC

Protein sequence (predicted transit sequence in red): MASQLRLHLAATPPLLPHRRPHLPRPLCPTLNPIRAPLPPLSRVLSHARPARAVGGGIEPKEGVVAEGDESGGGPVLVGEDSAAFELKDQSVASWAYFAGILGAVLVALNVLWIDPSTGVGTKFLDAVASVSDSHEVVMLLLTIIFAVVHSGMASLRESGEKIVGERVYRVLFAGISLPLAVTTIVYFINHRYDGTQLWQVQGITGIHELLWFSSFISFFFLYPSTFNLLEVAAVDKPKLHMWETGIMRITRHPQMVGQVIWCLAHTLWIGNSVAVAASVGLISHHLFGAWNGDRRLLSRYGEAFEVLKKRTSVMPFAAIIDGRQKLPKDYHKEFFRLPYVAITMLTLGAYFAHPLMQASSYQLPW

Supplementary Figure 9. Sequence of Maize Z-ISO codon optimized for E coli.

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-----------------E------------EE-----EE------HHHHHHHHHHHHHHHHHHHHHH---------HHEE Query: 47 HARPARAVGGGIEPKEGVVAEGDESGGGPVLVGEDSAAFELKDQSVASWAYFAGILGAVLVALNVLWIDPSTGVGTKFLD 126 Sbjct: 11 YLTRDMAWEPTYQDKKDIFPEEDFEGIKITDWSQWEDPFRL--TMDAYWKYQAEKEKKLYAIFDAFAQNNGHQN------ 82 -------------HHHH------------------------ HHHHHHHHHHHHHHHHHHHHHHHH------- EH-----HHHHHHHHHHHHHHHHH--HHHHHHHHHHHH-HHHHHHHHHHHHHHHHHHHHHHHHHH-----EE-----HHH Query: 127 AVASVSDSHEVVMLLLTIIFAVVHSGMASLRESGEKIVGERVYRVLFAGISLPLAVTTIVYFINHRYDGTQLWQVQGITG 206 Sbjct: 83 --ISDARYVNALKLFISGISPLEHAAFQGYSKVGRQFSGAGARVACQMQAIDELRHSQTQQHAMSHYNKH-FNGLHDGPH 159 -----HHHHHHHHHH-HHHHHHHHHHHHHHHHH---HHHHHHHHHHHHHHHHHHHHHHHHHHHHH--- -----HHHH HHHHHHHHHHHHHHHH-------HHHHH------------EEEE---HHHHHHHHHHHHHHHH---HHHHHHHHHHHHHH Query: 207 IHELLWFSSFISFFFLYPSTFNLLEVAAVDKPKLHMWETGIMRITRHPQMVGQVIWCLAHTLWIGNSVAVAASVGLISHH 286 Sbjct: 160 MHDRVWYLSVPKSFFDDARSAGPFEFLTAISFSFEYV------------LTNLLFVPFMSGAAYNGD-------MATVTF 220 H------HHHHHHHHHHHH--HHHHHHHH-------- HHHHHHHHHHHHH----H HHHHHH HHHHHHHHHHHHHH--HHHHHHHHH---HHHHEEE---------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH-- -- Query: 287 LFGAWNGDRRLLSRYGEAFEVLKKRTSVMPFAAIIDGRQKLPKDYHKEFFRLPYVAITMLTLGAYFAHPLMQASSY--QL 364 Sbjct: 221 GFSAQSDEARHMTLGLEVIKFILEQH----------------EDNVPIVQRWIDKWFWRGFRLLSLVSMMMDYMLPNKVM 284 HHHHHHHHHHHHHHHHHHHHHHHH-- -HHHHHHHHHHHHHHHHHHHHHHHHHHHHH-------- -- Query: 365 PW 366 Sbjct: 285 SW 286 HH

Supplementary Figure 10. Modeling a Z-ISO structure. The Z-ISO protein sequence from Zea mays was analyzed by the fold-recognition program, LOOPP which modeled Z-ISO onto Protein Databank structure 2INP. The alignment of the two sequences is shown (Query sequence is maize Z-ISO and the Subject (Sbjct) sequence is phenol hydroxylase; H=helical; E= extended).

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Control of carotenoid biosynthesis through a heme-based cis-trans isomeraseRESULTSExpression, isolation and activity assays of Z-ISOPredicting Z-ISO structure and localizationDetection of iron in Z-ISODetection of multiple heme species in Z-ISOZ-ISO has only one heme with reversible His and Cys ligandsDoes the heme ligand C263 function in isomerization?

DISCUSSIONMethodsONLINE METHODSGeneral gene cloning.Z-ISO expression and purification.Z-ISO in vitro enzyme assay.Bioinformatics.HPLC analysis.Z-ISO localization.Detection of metals in Z-ISO.Detection of heme.Binding of CN−.UV-visible spectroscopy Z-ISO difference spectra.Electron spin resonance spectroscopy.Magnetic circular dichroism.Site-directed mutagenesis and functional complementation in E. coli.Immunodetection of Z-ISO.

AcknowledgmentsCompeting financial interestsFigure 1 Z-ISO is an isomerase and integral membrane protein localized to chloroplasts.Figure 2 Z-ISO contains heme iron.Figure 3 EPR shows multiple heme species.Figure 4 MCD reveals redox-dependent changes in ligand coordination.Figure 5 Testing mutation of putative heme ligands on enzyme activity, heme binding and UV-vis spectrum of Z-ISO.Figure 6 Proposed mechanism of ligand rearrangement leading to active isomerization.

,DanaInfo=www.nature.com+nchembio.1840-S1.pdfE-mail: wurtzel@lehman.cuny.eduSupplementary Results

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