Abstract
Mg-protoporphyrin IX monomethyl ester (MgPME) cyclase catalyses the formation of the isocyclic ring, producing protochlorophyllide a and contributing substantially to the absorption properties of chlorophylls and bacteriochlorophylls. The O2-dependent cyclase is found in both oxygenic phototrophs and some purple bacteria. We overproduced the simplest form of the cyclase, AcsF, from Rubrivivax gelatinosus, in Escherichia coli. In biochemical assays the di-iron cluster within AcsF is reduced by ferredoxin furnished by NADPH and ferredoxin:NADP+ reductase, or by direct coupling to Photosystem I photochemistry, linking cyclase to the photosynthetic electron transport chain. Kinetic analyses yielded a turnover number of 0.9 min−1, a Michaelis–Menten constant of 7.0 µM for MgPME and a dissociation constant for MgPME of 0.16 µM. Mass spectrometry identified 131-hydroxy-MgPME and 131-keto-MgPME as cyclase reaction intermediates, revealing the steps that form the isocyclic ring and completing the work originated by Sam Granick in 1950.
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All supporting data are included in the Supplementary Information. The mass spectrometry raw data files used for Fig. 5 and Supplementary Fig. 3 have been deposited to https://figshare.shef.ac.uk/articles/dataset/13655774. Source data are provided with this paper.
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Acknowledgements
We thank D. Swainsbury, University of Sheffield, for providing membrane protein standards for gel filtration calibration and N. Soulier, The Pennsylvania State University, for supplying the BL21(DE3) ΔiscR strain. G.E.C., N.B.P.A. P.J.J. and C.N.H. thank the Biotechnology and Biological Sciences Research Council (BBSRC UK, award no. BB/M000265/1) for financial support. C.N.H. is also supported by European Research Council Synergy Award no. 854126. M.J.D. was supported by BBSRC UK (award no. BB/M012166/1).
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G.E.C., N.B.P.A., P.J.J., M.J.D. and C.N.H. designed the research. G.E.C., N.B.P.A. and P.J.J. performed research and analysed data. G.E.C., N.B.P.A., P.J.J., M.J.D. and C.N.H. wrote the manuscript.
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Extended data
Extended Data Fig. 1 Analysis of purified Anabaena Fd and FNR.
a, SDS-PAGE analysis of purified Anabaena Fd and FNR. b, Absorbance spectrum of purified Anabaena Fd. c, Absorbance spectrum of purified Anabaena FNR.
Extended Data Fig. 2 HPLC elution profiles of pigment extracts from coupled PSI-cyclase assays.
A complete assay contained 2 µM AcsF, 0.04 mg ml−1 spinach Fd, 14 µM MgPME, spinach PSI containing 6 (1× PSI) or 22.4 µM (~4× PSI) Chl a, 20 µM spinach Pc, 2 mM Asc, 60 µM DCPIP and 0.29 mg ml−1 catalase. Assays were incubated either in the dark for 30 min, or under red light illumination for 15 or 30 min. Pigment extracts from the assays were analysed by HPLC and pigment elution was monitored by fluorescence at 640 nm excited at 440 nm. Pigment species were identified by retention times and fluorescence spectra (as in Fig. 4). See Supplementary Fig. 4a for HPLC analysis of pigment extracts from additional control assays.
Extended Data Fig. 3 The diiron binding motif and proposed diiron ligation of AcsF.
a, Sequence alignments showing the conserved diiron binding motif of AcsF proteins. Sequences are from Synechocystis sp. PCC 6803 (CycI, BAA16583), Arabidopsis thaliana (CHL27, NP_191253), Chlamydomonas reinhardtii (CRD1, XP_001692557; CTH1, XP_001691047), Rubrivivax gelatinosus IL144 (AcsF, BAL96694) and Rhodobacter sphaeroides 2.4.1 (0294, abbreviated for RSP_0294, YP_353369). Conserved, highly similar and similar residues are marked with asterisks, colons and full stops, respectively. The putative diiron ligands are in red and bold. Full-length protein sequences were used for alignments but for clarity, only the putative diiron binding motifs with the residue range indicated, are shown. b, Sequence homologies between the diiron binding motifs of AcsF proteins and the soluble methane monooxygenase hydroxylase subunit from Methylococcus capsulatus Bath (MMOH, P22869). c, Proposed coordination of the diiron ligands of AcsF at the diferrous state based on the crystal structure of MMOH (PDB, 1FYZ)51.
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Chen, G.E., Adams, N.B.P., Jackson, P.J. et al. How the O2-dependent Mg-protoporphyrin monomethyl ester cyclase forms the fifth ring of chlorophylls. Nat. Plants 7, 365–375 (2021). https://doi.org/10.1038/s41477-021-00876-3
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DOI: https://doi.org/10.1038/s41477-021-00876-3
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