Swapping metals in Fe- and Mn-dependent dioxygenases: Evidence for oxygen activation without a change in metal redox state
- Joseph P. Emerson*,†,
- Elena G. Kovaleva†,‡,
- Erik R. Farquhar*,†,
- John D. Lipscomb†,‡,§, and
- Lawrence Que, Jr.*,†,¶
- *Department of Chemistry,
- ‡Department of Biochemistry, Molecular Biology, and Biophysics, and
- †Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, MN 55455
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Edited by J. Martin Bollinger, Pennsylvania State University, University Park, PA, and accepted by the Editorial Board March 3, 2008 (received for review November 26, 2007)
Abstract
Biological O2 activation often occurs after binding to a reduced metal [e.g., M(II)] in an enzyme active site. Subsequent M(II)-to-O2 electron transfer results in a reactive M(III)-superoxo species. For the extradiol aromatic ring-cleaving dioxygenases, we have proposed a different model where an electron is transferred from substrate to O2 via the M(II) center to which they are both bound, thereby obviating the need for an integral change in metal redox state. This model is tested by using homoprotocatechuate 2,3-dioxygenases from Brevibacterium fuscum (Fe-HPCD) and Arthrobacter globiformis (Mn-MndD) that share high sequence identity and very similar structures. Despite these similarities, Fe-HPCD binds Fe(II) whereas Mn-MndD incorporates Mn(II). Methods are described to incorporate the nonphysiological metal into each enzyme (Mn-HPCD and Fe-MndD). The x-ray crystal structure of Mn-HPCD at 1.7 Å is found to be indistinguishable from that of Fe-HPCD, while EPR studies show that the Mn(II) sites of Mn-MndD and Mn-HPCD, and the Fe(II) sites of the NO complexes of Fe-HPCD and Fe-MndD, are very similar. The uniform metal site structures of these enzymes suggest that extradiol dioxygenases cannot differentially compensate for the 0.7-V gap in the redox potentials of free iron and manganese. Nonetheless, all four enzymes exhibit nearly the same K M and V max values. These enzymes constitute an unusual pair of metallo-oxygenases that remain fully active after a metal swap, implicating a different way by which metals are used to promote oxygen activation without an integral change in metal redox state.
Footnotes
- §To whom correspondence may be addressed at: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 321 Church Street SE, Minneapolis, MN 55455. E-mail: lipsc001{at}umn.edu
- ¶To whom correspondence may be addressed at: Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, MN 55455. E-mail: larryque{at}umn.edu
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Author contributions: J.P.E., J.D.L., and L.Q. designed research; J.P.E., E.G.K., and E.R.F. performed research; J.P.E., E.G.K., and E.R.F. analyzed data; and J.P.E., E.R.F., J.D.L., and L.Q. wrote the paper.
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The authors declare no conflict of interest.
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This article is a PNAS Direct Submission. J.M.B. is a guest editor invited by the Editorial Board.
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Data deposition: The x-ray crystal structure of Mn-HPCD has been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3BZA).
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See Commentary on page 7341.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0711179105/DCSupplemental.
- © 2008 by The National Academy of Sciences of the USA
