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Protein Science (2002), 11:2464-2470.
Copyright © 2002 The Protein Society

Zinc-substituted Desulfovibrio gigas desulforedoxins: Resolving subunit degeneracy with nonsymmetric pseudocontact shifts

Brian J. Goodfellow1, Sofia G. Nunes2, Frank Rusnak3, Isabel Moura2, Carla Ascenso2, José J.G. Moura2, Brian F. Volkman4 and John L. Markley5

1 Departamento de Química, Universidade de Aveiro, 3810-193 Aveiro, Portugal
2 Departamento de Química, Centro de Química Fina e Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2825-114 Monte de Caparica, Portugal
3 Section of Hematology Research and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, 55905 USA
4 Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA
5 National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin 53706, USA

Reprint requests to: John L. Markley, National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin–Madison, 433 Babcock Drive, Madison, WI 53706, USA; e-mail: markley{at}nmrfam.wisc.edu; fax: (608) 262-3759; Brian F. Volkman, Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, USA; e-mail: bvolkman{at}mcw.edu; fax: (414) 456-6510; Brian J. Goodfellow, Departamento de Química, Universidade de Aveiro, 3810–193 Aveiro, Portugal; e-mail: brian.goodfellow{at}dq.ua.pt; fax: 351-234-370084.

Desulfovibrio gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe-4S] center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the desulforedoxins containing iron can be reduced with added dithionite. For NMR studies, different protein samples were prepared labeled with 15N or 15N + 13C. Spectral assignments were determined for [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx from 3D 15N TOCSY-HSQC and NOESY-HSQC data, and compared with those reported previously for [Zn(II)/Zn(II)]Dx. Assignments for the 13C{alpha} shifts were obtained from an HNCA experiment. Comparison of 1H–15N HSQC spectra of [Zn(II)/Zn(II)]Dx, [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx revealed that the pseudocontact shifts in [Fe(II)/Zn(II)]Dx can be decomposed into inter- and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for [Fe(II)/Fe(II)]Dx. The degree of linearity observed in the pseudocontact shifts for residues >=8.5 Å from the metal center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx. The results suggest a general strategy for the analysis of NMR spectra of homo-oligomeric proteins in which a paramagnetic center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits.

Keywords: NMR; pseudocontact shifts; desulforedoxin; [Fe-4S] center; paramagnetic protein; Desulfovibrio gigas

Abbreviations: Cp, Clostridium pasteurianum • Dg, Desulfovibrio gigas • Dx, desulforedoxin from Desulfovibrio gigas • Rd, rubredoxin • HSQC, heteronuclear single quantum coherence • NOESY, nuclear Overhauser effect spectroscopy • TOCSY, total correlation spectroscopy


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Copyright © 2002 by The Protein Society.