Construction of recombinant plasmids

Coding sequences of full-length Synechocystis sp. PCC6803 Hik2 (slr1147), A. thaliana CSK (At1g67840), and P. tricornutum CSK (PHATRDRAFT_41268) were amplified by using the primer pairs listed in Supplementary Table 3. PCR products of Hik2 and Arabidopsis CSK were digested with NdeI and XhoI endonucleases (New England BioLabs) and cloned into pET-21b (Novagen) expression vector digested with the same enzymes. The Phaeodactylum CSK PCR product was digested with KpnI and XhoI and cloned into pETG-41A (EMBL) expression vector. The identities of the recombinant clones were confirmed by sequencing.

Site-directed mutagenesis of conserved cysteines

Mutagenesis of the conserved cysteine residues of the Synechocystis Hik2 (C19, C35, and C153) to serines were made using the Stratagene QuickChange Site-directed Mutagenesis Kit. The primer pairs used are listed in Supplementary Table 3. Mutagenesis was confirmed by sequencing.

Expression and purification of recombinant CSK

The BL21 (DE3) Rosetta chemically competent cells (Stratagene) were transformed with CSK expression constructs. Transformed bacterial colonies, grown on agar plates, were used to inoculate starter cultures (10 mL each) in Luria Broth (LB) growth media with 100 μg mL−1 ampicillin. Each culture was grown overnight, then diluted 1:100 in 1 L LB media and grown at 37 °C to an optical density at 600 nm of ~0.55, before inducing protein expression with 0.5 mM IPTG (Fisher Scientific). Bacterial cultures were grown for an additional 16 h at 16 °C. Cells were harvested by centrifugation at 6000 rpm for 10 min at 4 °C, and the pellet re-suspended in buffer containing 300 mM NaCl, 20 mM Tris-HCl, pH 8.0, 25 mM imidazole, 5 mM DTT, and 1 mM PMSF. The cells were lysed with an EmulsiFlex-C3 homogenizer (Avestin). The lysate was clarified by centrifugation at 35,000 × g for 20 min at 4 °C. The supernatant was applied to a HisTrapTM HP 5 mL Ni2+-charged affinity chromatography column (GE Healthcare Life Sciences), and the recombinant protein was purified according to the manufacturer’s instructions. The purified proteins were buffer exchanged into 20 mM Tris-HCl, pH 8, and 100 mM NaCl before further analysis.

In vitro reconstitution of the Fe-S cluster

The reconstitution procedure was carried out in an anaerobic glove bag (Glas-Col) filled with argon gas. The desalted Syn-CSK, Ara-CSK, or Phaeo-CSK proteins at a concentration of 8.4 mg mL−1 were reduced with 10 mM DTT anaerobically and incubated for 30 min on ice. After this pretreatment, equal amounts of ferrous ammonium sulfate and sodium sulfide were slowly added at a stoichiometry of 15:1 (iron or sulfide to protein) and incubated at 22 °C for 3.5 h. Precipitant was removed by centrifugation at 5000 × g at 4 °C for 5 min and the unbound iron and sulfide removed by buffer exchange with 2 mM DTT, 100 mM NaCl, and 20 mM Tris-HCl, pH 8.0 using a PD-10 column.

Acid-labile sulfide and iron quantification

Acid-labile sulfide content was measured as in ref. 40 by formation of methylene blue (absorbance maximum at 666 nm) from the reaction of N,N-dimethyl-p-phenylenediamine with H 2 S and excess FeCl 3 . The same method was downscaled 12-fold for determination of sulfide in 96-well plates (200 μL sample volume) and confirmation by visible spectroscopy that methylene blue was produced. Standardization was carried out with freshly purchased Na 2 S. This method is highly specific for acid-labile sulfide and does not give a response with commonly encountered sulfur compounds, including protein-bound cysteine or methionine41. Iron was quantified by using the commercial Quantichrom Iron Assay Kit purchased from Bioassay Systems. Protein concentrations for assays and Fe/S analysis were determined by the Bradford method, using bovine serum albumin as a standard.

UV-Vis and EPR spectroscopy

UV-Vis absorbance spectra were recorded at room temperature with a HITACHI U-3900H spectrophotometer. The EPR spectrum was recorded with a Bruker ELEXSYS E580 spectrometer, operating at X-band, and the temperature controlled using an Oxford ITC503 cryostat system. Experiments were set to achieve maximum signal-to-noise ratio without producing saturation and/or distortion of EPR signals. The EPR parameters were: microwave power, 10 mW; modulation amplitude, 10 G; modulation frequency, 100 kHz at 5–45 K. Reduced and oxidized samples of the protein (Syn-CSK 173 µM, Phaeo-CSK 115 µM, and Ara-CSK 176 µM) were prepared in a buffer containing 2 mM DTT, 100 mM NaCl, and 20 mM Tris-HCl, pH 8.0. The oxidized protein was obtained by incubation in air at 22 °C for 5 min and reduced protein by addition of 5 mM dithionite dissolved in 1 M Tris-HCl, pH 8, followed by incubation at 22 °C for 5 min. Two hundred microliters of oxidized or reduced samples, taken in quartz tubes, were immediately flash frozen in liquid nitrogen for EPR analysis.

Anion exchange chromatography

The reconstituted Syn-CSK was first desalted into 10 mM NaCl and 25 mM HEPES, pH 8 with a PD10 column, before being subjected to DEAE-Sepharose CL6B IEC (Sigma-Aldrich). The ion exchange column was washed with 2 column volume of equilibration buffer and the protein was eluted with 5 column volume 300 mM NaCl and 25 mM HEPES, pH 8. Two hundred microliters of the concentrated eluate was taken in quartz tubes and immediately flash frozen in liquid nitrogen for EPR analysis.

X-ray absorption spectra (XAS) and EXAFS measurements

XAS were collected at the Advanced Photon Source (APS) at Argonne National Laboratory on bending magnet beamline 20 having electron energy 7 GeV and average current of 100 mA. The radiation was monochromatized by a Si(110) crystal monochromator. The intensity of the X-rays was monitored by three ion chambers (I 0 , I 1 , and I 2 ) filled with 30% nitrogen and 70% helium and placed before the sample (I 0 ) and after the sample (I 1 and I 2 ). Iron foil was placed between I 1 and I 2 and its absorption was recorded with each scan for energy calibration. In all, 1.2 mmol of Syn-CSK protein was prepared in a reaction buffer containing 0.1 M NaCl and 0.02 M Tris-HCl (pH 8.0). Plastic (Lexan) EXAFS sample holders (inner dimensions of 12 mm × 2 mm × 3 mm) filled with frozen samples were inserted into a cryostat pre-cooled to 20 K. The samples were kept at 20 K in a He atmosphere at ambient pressure. Data were recorded as fluorescence excitation spectra using a 13-element energy-resolving detector. In order to reduce the risk of sample damage by X-ray, defocused mode (beam size 1 × 1.6 mm) was used and no damage was observed. The shutter was synchronized with the “scan software” preventing exposure to X-rays in between scans. Fe XAS energy was calibrated by the maximum of the pre-edge feature of the iron foil (7112 eV), which was placed between I 1 and I 2 ionization chambers. EXAFS scans with 5 eV steps in the pre-edge region, 0.5 eV steps through the edge, and 0.05 Å–1 steps from k = 2.0 to 14 Å–1 were used.

Athena software was used for data processing42. The energy scale for each scan was normalized using Fe foil standard and multiple scans for the same samples were added together. The data in energy space were pre-edge corrected, normalized, and background corrected. The processed data were then converted to the photoelectron wave vector (k) space and weighted by k3. The electron wave number is defined as \(k = [2m(E - E_0)/\hbar ^2]^{1/2}\), where E 0 is the energy origin or the threshold energy. K-space data were truncated near the zero crossings (k = 4.0–10.0 Å−1) before the Fourier transformation in R-space. The k-space data were transferred into the Artemis Software for curve fitting. In order to fit the data, the Fourier peaks were isolated by applying a Hanning window to the first and last 15% of the chosen range, leaving the middle 70% untouched. Curve fitting was performed using ab initio-calculated phases and amplitudes from the FEFF9 program from the University of Washington. Ab initio-calculated phases and amplitudes were used in the EXAFS equation43:

$$\chi (k) = S_0^2\mathop {\sum }\limits_j \frac{{N_j}}{{kR_j^2}}f_{{\rm{eff}}_j}(\pi ,k,R_j)e^{ - 2\sigma _j^2k^2}e^{\frac{{ - 2R_j}}{{\lambda _j(k)}}}{\mathrm{sin}}(2kR_j + \emptyset _{ij}(k))$$ (1)

where N j is the number of atoms in the jth shell; R j the mean distance between the absorbing atom and the atoms in the jth shell; f eff j (π, k, R j ) is the ab initio amplitude function for shell j, and the Debye–Waller term \(e^{ - 2\sigma _j^2k^2}\) accounts for damping due to static and thermal disorder in absorber–backscatterer distances. The mean free path term \(e^{\frac{{ - 2R_j}}{{\lambda _j(k)}}}\) reflects losses due to inelastic scattering, where λ j (k) is the electron mean free path. The oscillations in the EXAFS spectrum are reflected in the sinusoidal term \(\sin (2kR_j + \emptyset _{ij}(k))\), where \(\emptyset _{ij}(k)\) is the ab initio phase function for shell j. This sinusoidal term shows the direct relation between the frequency of the EXAFS oscillations in k-space and the absorber–backscatterer distance. S 0 2 is an amplitude reduction factor representing central atom shake-up and shake-off effects. The mean free path of the electron (λ) is due to the finite core hole lifetime and interactions with the valence electrons.

The EXAFS equation (Eq. 1) was used to fit the experimental data using N, S o 2, E 0 , R, and σ2 as variable parameters (see fit results in Table 1). N refers to the number of coordination atoms surrounding Fe in each shell. The quality of fit was evaluated by the R-factor and the reduced χ2 value. R-factor is used to denote closeness of fit. An R-factor <2% denotes that the fit is good enough, whereas R-factor between 2% and 5% denotes that the fit is correct within a consistently broad model44. The reduced χ2 value is used to compare fits as more absorber–backscatter shells are included to fit the data. Reduced χ2 value justifies the inclusion of an additional shell.

Potentiometric redox titration

The protocol followed was modified from refs. 45,46. Five milliliters of 8.4 mg mL−1 Syn-CSK protein was prepared in a reaction buffer containing 0.1 M NaCl, 0.05 M Tris-HCl (pH 8.0), and 10% glycerol. Forty micromolar of each of the following mediators: 1,4-benzoquinone (E m7 = + 280 mV), 1,2-naphthoquinone (E m7 = +135 mV), 1,4-naphthoquinone (E m7 = + 65 mV), duroquinone (E m7 = +7 mV), 2,5-dihydroxy-1,4-benzoquinone (E m7 = −60 mV), anthraquinone-2,6-disulfonic acid disodium salt (E m7 = −184 mV), and sodium anthraquinone-2-sulfonate (E m7 = −225 mV), were added to the protein solution and allowed to equilibrate for 30 min before recording potential. The potential was gradually lowered by adding substochiometric amount of dithionite dissolved in argon-bubbled 1 M Tris-HCl (pH 8). Two hundred microliters of CSK protein sample, poised at a desired potential, was withdrawn with syringe and was injected into an EPR tube and frozen in liquid nitrogen for EPR analysis. Anaerobic conditions were maintained by flushing the cuvette with water-saturated argon. The redox electrode system was calibrated with saturated quinhydrone dissolved in 0.1 M sodium phosphate, pH 7. The accuracy of the E m measurement was checked at the negative potential by titration of 20 µM FMN, which has a midpoint potential of −205 mV at pH 7.0. The midpoint potential was calculated using a derivation of the Nernst equation:

$${\mathrm{EPR}}\,{\mathrm{signal}} = \frac{{{\mathrm{Maximum}}\,{\mathrm{EPR}}\,{\mathrm{signal}}}}{{1 + e^{(E_{\mathrm{m}} - E)\frac{F}{{RT}}}}}$$

where E = potential in voltage,

E m = midpoint potential in voltage,

F = Faraday constant = 96,480 C mol−1,

R = Gas constant = 8.314 J K−1 mol−1, and

T = Temperature in Kelvin.

In vitro autophosphorylation

Autophosphorylation was carried out in a final reaction volume of 25 µL. The reaction contained 20 µL of 2 µM reconstituted Syn-CSK, which was desalted into 1.25-fold concentrated kinase reaction buffer containing 62.5 mM Tris-HCl at pH 8, 6.25 mM KCl, 12.5% glycerol, and 6.25 mM MgCl 2 . Where indicated, 1 µL redox reagent was added to the protein, with the specified final concentrations: 1 mM NADPH (dissolved in water), 0.5 mM sodium dithionite (dissolved in 1 M Tris-HCl, pH 8), 0.5 mM duroquinone (dissolved in 95% ethanol), 0.5 mM duroquinol (dissolved in 95% ethanol), 0.5 mM decyl-PQ (dissolved in 95% ethanol), and 0.5 mM decyl-plastoquinol (dissolved in 95% ethanol). The reduced quinones were prepared by bubbling oxidized quinones with hydrogen gas in the presence of a platinum catalyst. Spinach ferredoxin (Sigma-Aldrich) was first reduced with 2 mM dithionite in a glove bag for 5 min and desalted into kinase reaction buffer lacking dithionite. Six micrograms of the reduced and desalted ferredoxin was then added to Syn-CSK. The kinase reaction tube was covered with 5 µL of mineral oil in order to keep the gas phase at a minimum and incubated at 22 °C for 5 min. The autophosphorylation reaction was initiated by the addition of 4 µL of a 6.25-fold concentrated ATP solution containing 2.5 mM disodium ATP (Sigma) and 2.5 µCi [γ-32P]ATP (6000 Ci mmol−1) (PerkinElmer). Reactions were incubated for 60 s at 22 °C, and the autophosphorylation reaction terminated by the addition of 6.25 µL of 5-fold concentrated Laemmli sample buffer47. Reaction products were separated by sodium dodecyl sulfate–6 M urea–11% (w/v) polyacrylamide gel electrophoresis. The gel was rinsed with gel running buffer and subsequently exposed to a phosphor plate overnight. The incorporated γ-32P was visualized by autoradiography.

Immunopurification and phosphopepide identification of CSK

Six-week-old Arabidopsis plants, containing the HA-FLAG-tagged CSK, were transferred to far-red light. Chloroplasts were isolated from the illuminated plants and the CSK protein was purified by the FLAG-HA Tandem Affinity Purification Kit (Sigma-Aldrich). The purified proteins were subjected to trypsin digestion, phosphopeptide enrichment18, and LC-MS/MS analysis.

Size exclusion chromatography

The oligomeric state of Syn-CSK was determined by subjecting the purified protein to Superdex 200 10/300GL Increase (GE Healthcare Life Sciences) size exclusion chromatography, equilibrated with 20 mM Tris-HCl (pH 8.0) and 20 mM NaCl. The molecular mass of CSK was determined by using the calibration curve at 20 mM NaCl and 20 mM Tris-HCl (pH 8.0) (Supplementary Fig. 7).

Chemical crosslinking

Syn-CSK protein was desalted into crosslinking reaction buffer (25 mM HEPES-NaOH at pH 8.0, 5 mM KCl, and 5 mM MgCl 2 ) using a PD-10 desalting column. Chemical crosslinking was carried out in a total reaction volume of 1.0 mL containing 4 µM of Syn-CSK protein. The Syn-CSK was treated with 0.5 mM dithionite under anaerobic condition or air oxidized for 5 min. The crosslinking agent disuccinimidyl suberate (DSS) was added from a 24.7 mM stock solution in dimethyl sulfoxide to give a final DSS concentration of 2 mM. Reactions were incubated at 22 °C for 30 min. Reactions were stopped by addition of a solution containing 50 mM Tris-HCl and 10 mM glycine, pH 7.5.

Far-UV CD analysis of thermal stability

Far-UV CD spectra and structure stability were measured in stirred quartz cuvettes with an optical path length of 1 cm using a Chirascan spectropolarimeter (Applied Photophysics) in 20 mM Tris, pH 7.8, and 0.1 NaCl. Thermal denaturation profiles were obtained by measuring the amplitude of the CD signal at 222 nm over a temperature range of 20–80 °C with a rate of temperature increase of 1 °C min−1 and step of 0.4 °C. Changes in protein thermal stability caused by oxidation by 50 µM K 3 Fe(CN) 6 were detected by a comparison of peak positions of the first derivative of the CD amplitude versus thermal melting profiles.

Quantitative real-time PCR

Total RNA was isolated from the leaves of 12-day-old seedlings using the ZR Plant RNA MiniPrep Kit (Zymo Research). RNA was treated with RNase-free DNase (Zymo Research) to eliminate possible DNA contamination. First-strand cDNA was synthesized from 0.400 μg of RNA with the RevertAid First Strand cDNA Synthesis Kit (Fisher Scientific). Real-time quantitative reverse transcriptase polymerase chain reaction was performed with a one-step QuantiNova SRBR Green PCR Kit (Qiagen) in a StepOnePlus thermocycler (Applied Biosystems). The expression values of target genes were normalized to both total RNA and endogenous Actin8 control. The relative changes in gene expression were analyzed by 2−ΔΔCt method.

Statistics and reproducibility

Statistical significance of data were tested by unpaired Student’s t test. The sample size (n) and the nature of replicates have been given wherever relevant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.