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X-ray crystal structure of the light-independent protochlorophyllide reductase

Nature volume 465, pages 110–114 (2010)Cite this article

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Abstract

Photosynthetic organisms adopt two different strategies for the reduction of the C17 = C18 double bond of protochlorophyllide (Pchlide) to form chlorophyllide a, the direct precursor of chlorophyll a (refs 1–4). The first involves the activity of the light-dependent Pchlide oxidoreductase5,6,7,8,9, and the second involves the light-independent (dark-operative) Pchlide oxidoreductase10 (DPOR). DPOR is a nitrogenase-like enzyme consisting of two components, L-protein (a BchL dimer) and NB-protein (a BchN–BchB heterotetramer), which are structurally related to nitrogenase Fe protein and MoFe protein, respectively10,11. Here we report the crystal structure of the NB-protein of DPOR from Rhodobacter capsulatus at a resolution of 2.3 Å. As expected, the overall structure is similar to that of nitrogenase MoFe protein: each catalytic BchN–BchB unit contains one Pchlide and one iron–sulphur cluster (NB-cluster) coordinated uniquely by one aspartate and three cysteines. Unique aspartate ligation is not necessarily needed for the cluster assembly but is essential for the catalytic activity. Specific Pchlide-binding accompanies the partial unwinding of an α-helix that belongs to the next catalytic BchN–BchB unit. We propose a unique trans-specific reduction mechanism in which the distorted C17-propionate of Pchlide and an aspartate from BchB serve as proton donors for C18 and C17 of Pchlide, respectively. Intriguingly, the spatial arrangement of the NB-cluster and Pchlide is almost identical to that of the P-cluster and FeMo-cofactor in nitrogenase MoFe-protein, illustrating that a common architecture exists to reduce chemically stable multibonds of porphyrin and dinitrogen.

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Figure 1: Pchlide reduction and crystal structures of DPOR NB-protein and nitrogenase MoFe protein.
Figure 2: Structures around the NB-cluster and the activity of NB-protein mutants.
Figure 3: Pchlide-binding cavity and a proposed reaction mechanism for trans -specific reduction.
Figure 4: Common architecture of DPOR and nitrogenase.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors for the structures reported here are available from the Protein Data Bank with accession codes of 3AEK (Pchlide-bound form), 3AEQ (anaerobic Pchlide-bound form), 3AER (Pchlide-free form), 3AES (selenomethionine-substituted Pchlide-free form), 3AET (D36C form) and 3AEU (D36A form).

Change history

  • 06 May 2010

    The position of the Mg in Fig. 3d was corrected.

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Acknowledgements

We thank K. Fukuyama, T. Omata and K. Terauchi for discussions; J. Peters and R. Cogdell for a critical reading of the manuscript; H. Yamamoto for establishing the functional overexpression system in E. coli; J. Harimoto for technical help in the sequence confirmation of a series of expression vectors; C. E. Bauer for donating the broad host-range vector pBBR1MSC2 and the triparental mating system for R. capsulatus; and the staff at the Photon Factory, KEK, Japan, for support during data collection. This work was supported by Grants-in-Aid for Scientific Research numbers 17687008 and 18054308 (G.K.), 19570036, 14COEA2 and 20200063 (Y.F.), 19750148 (T.M.) and 19350088 (H.T.) from the Japan Society for the Promotion of Science (JSPS), Precursory Research for Embryonic Science and Technology of the Japan Science and Technology Agency (JST) (Y.F.), the Kato Memorial Science Foundation (G.K.), the Toyoaki Scholarship Foundation and the Japan Securities Scholarship Foundation (Y.F.). N.M and J.N. grateful for fellowships from the JSPS for Japanese Junior Scientists (numbers 19010733 and 2110614).

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Author notes

  1. Tomoo Shiba

    Present address: Present address: Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.,

  2. Norifumi Muraki and Jiro Nomata: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Life Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan,

    Norifumi Muraki & Tomoo Shiba

  2. Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan,

    Norifumi Muraki & Genji Kurisu

  3. Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan,

    Jiro Nomata, Kozue Ebata & Yuichi Fujita

  4. Department of Bioscience and Biotechnology, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan,

    Tadashi Mizoguchi & Hitoshi Tamiaki

  5. Presto, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan,

    Yuichi Fujita

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Contributions

Purification of NB-protein and site-directed mutagenesis were conducted by J.N., K.E. and N.M.; the enzymatic assay of NB-protein was conducted by J.N.; the crystal structures of NB-protein were solved by N.M., T.S. and G.K.; chlorophyll c was prepared by T.M. and H.T.; G.K. and Y.F. contributed to the design of the experiments and writing the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Genji Kurisu or Yuichi Fujita.

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The authors declare no competing financial interests.

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This file contains Supplementary Figures 1-6 with legends, Supplementary Tables 1-2, a Supplementary Discussion and Supplementary References. (PDF 8590 kb)

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Muraki, N., Nomata, J., Ebata, K. et al. X-ray crystal structure of the light-independent protochlorophyllide reductase. Nature 465, 110–114 (2010). https://doi.org/10.1038/nature08950

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