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Crystal structures of ferredoxin variants exhibiting large changes in [Fe–S] reduction potential

Nature Structural Biology volume 9, pages 188–192 (2002)Cite this article

Abstract

Elucidating how proteins control the reduction potentials (E0′) of [Fe–S] clusters is a longstanding fundamental problem in bioinorganic chemistry. Two site-directed variants of Azotobacter vinelandii ferredoxin I (FdI) that show large shifts in [Fe–S] cluster E0′ (100–200 mV versus standard hydrogen electrode (SHE)) have been characterized. High resolution X-ray structures of F2H and F25H variants in their oxidized forms, and circular dichroism (CD) and electron paramagnetic resonance (EPR) of the reduced forms indicate that the overall structure is not affected by the mutations and reveal that there is no increase in solvent accessibility nor any reorientation of backbone amide dipoles or NH–S bonds. The structures, combined with detailed investigation of the variation of E0′ with pH and temperature, show that the largest increases in E0′ result from the introduction of positive charge due to protonation of the introduced His residues. The smaller (50–100 mV) increases observed for the neutral form are proposed to occur by directing a Hδ+–Nδ− dipole toward the reduced form of the cluster.

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Figure 1: Crystal structures of FdI mutants showing His 2 and His 25 contacts, solvent accessibility and electron density.
Figure 2: UV/Vis and EPR spectra of FdI variants.
Figure 3: The pH dependence of E0′ values for [[3Fe–4S](Cys3)]2−/3− and [[4Fe–4S](Cys4)]2−/3− centers in F2H and F25H variants of FdI.
Figure 4: Protonation of His 25, not of the cluster, causes the pH dependence of reduction potential of the [[3Fe–4S](Cys3)]2−/3− center in F25H.

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Acknowledgements

The authors wish to acknowledge V. Roberts and L. Noodleman at The Scripps Research Institute, and M. Gunner, City College, New York, and J. Hirst, MRC, Cambridge for helpful discussions. G. Murcott (Oxford) assisted in some of the experiments. We also thank T.L. Poulos and H. Li at University of California-Irvine for use of laboratory facilities for X-ray crystallography. This work was funded by grants from UK EPSRC and BBSRC to F.A.A. and NIH grants to C.D.S. and B.K.B.

Barbara K. Burgess died unexpectedly on December 30, 2001. We would like to pay tribute to her many contributions to science; in particular her studies of the proteins and cofactors involved in biological nitrogen fixation, and her elucidation of many important properties of iron-sulfur clusters.

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Author notes

  1. Kaisheng Chen

    Present address: The Genomics Institute of the Novartis Research Foundation, 3115 Merryfield Row, Suite 200, San Diego, California, 92121, USA

  2. Kaisheng Chen, Christopher A. Bonagura, Gareth J. Tilley, James P. McEvoy and Fraser A. Armstrong: These authors contributed equally to the work.

Authors and Affiliations

  1. Department of Molecular Biology and Biochemistry, University of California, Irvine, 92697, California, USA

    Kaisheng Chen, Christopher A. Bonagura, Yean-Sung Jung & Barbara K. Burgess

  2. Department of Chemistry, Oxford University, Oxford, OX1 3QR, UK

    Gareth J. Tilley, James P. McEvoy & Fraser A. Armstrong

  3. Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, 92037, California, USA

    C. David Stout

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Correspondence should be addressed to Susan Bryant. email: svbryant@uci.edu

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Chen, K., Bonagura, C., Tilley, G. et al. Crystal structures of ferredoxin variants exhibiting large changes in [Fe–S] reduction potential. Nat Struct Mol Biol 9, 188–192 (2002). https://doi.org/10.1038/nsb751

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