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A productive NADP+ binding mode of ferredoxin–NADP+ reductase revealed by protein engineering and crystallographic studies

Nature Structural Biology volume 6, pages 847–853 (1999)Cite this article

Abstract

The flavoenzyme ferredoxin–NADP+ reductase (FNR) catalyzes the production of NADPH during photosynthesis. Whereas the structures of FNRs from spinach leaf and a cyanobacterium as well as many of their homologs have been solved, none of these studies has yielded a productive geometry of the flavin–nicotinamide interaction. Here, we show that this failure occurs because nicotinamide binding to wild type FNR involves the energetically unfavorable displacement of the C-terminal Tyr side chain. We used mutants of this residue (Tyr 308) of pea FNR to obtain the structures of productive NADP+ and NADPH complexes. These structures reveal a unique NADP+ binding mode in which the nicotinamide ring is not parallel to the flavin isoalloxazine ring, but lies against it at an angle of ~30°, with the C4 atom 3 Å from the flavin N5 atom.

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Figure 1: Spectroscopic characterization of wild type, Y308S and Y308W pea FNRs.
Figure 2: NADP+ binding mode in Y308S–NADP+ complex.
Figure 3: NADP(H) binding to pea FNR mutants.

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Acknowledgements

We thank L. Piubelli for performing some of the spectroscopic experiments, S.E. Ealick for the use of his area detector facility, and T.P. Begley and V. Massey for helpful discussions. This work was supported by grants from the NSF to P.A.K., from CONICET and FONCYT (Argentina) to E.A.C., and from MURST to G.Z. N.C. was a recipient of a John Simon Guggenheim Fellowship.

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Authors and Affiliations

  1. Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, 14853 , New York, USA

    Zhan Deng & P. Andrew Karplus

  2. Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Via Celoria 26, Milano, 20133, Italy

    Alessandro Aliverti & Giuliana Zanetti

  3. Molecular Biology Division, PROMUBIE, CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario , 2000, Argentina

    Adrián K. Arakaki, Jorgelina Ottado, Elena G. Orellano, Nora B. Calcaterra, Eduardo A. Ceccarelli & Néstor Carrillo

  4. Department of Biochemistry and Biophysics, Oregon State University, Corvallis, 97331, Oregon, USA

    P. Andrew Karplus

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  1. Zhan Deng
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Correspondence to P. Andrew Karplus.

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Deng, Z., Aliverti, A., Zanetti, G. et al. A productive NADP+ binding mode of ferredoxin–NADP+ reductase revealed by protein engineering and crystallographic studies. Nat Struct Mol Biol 6, 847–853 (1999). https://doi.org/10.1038/12307

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