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Probing the role of the divalent metal ion in uteroferrin using metal ion replacement and a comparison to isostructural biomimetics

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Abstract

Purple acid phosphatases (PAPs) are a group of heterovalent binuclear metalloenzymes that catalyze the hydrolysis of phosphomonoesters at acidic to neutral pH. While the metal ions are essential for catalysis, their precise roles are not fully understood. Here, the Fe(III)Ni(II) derivative of pig PAP (uteroferrin) was generated and its properties were compared with those of the native Fe(III)Fe(II) enzyme. The k cat of the Fe(III)Ni(II) derivative (approximately 60 s−1) is approximately 20% of that of native uteroferrin, and the Ni(II) uptake is considerably faster than the reconstitution of full enzymatic activity, suggesting a slow conformational change is required to attain optimal reactivity. An analysis of the pH dependence of the catalytic properties of Fe(III)Ni(II) uteroferrin indicates that the μ-hydroxide is the likely nucleophile. Thus, the Ni(II) derivative employs a mechanism similar to that proposed for the Ga(III)Zn(II) derivative of uteroferrin, but different from that of the native enzyme, which uses a terminal Fe(III)-bound nucleophile to initiate catalysis. Binuclear Fe(III)Ni(II) biomimetics with coordination environments similar to the coordination environment of uteroferrin were generated to provide both experimental benchmarks (structural and spectroscopic) and further insight into the catalytic mechanism of hydrolysis. The data are consistent with a reaction mechanism employing an Fe(III)-bound terminal hydroxide as a nucleophile, similar to that proposed for native uteroferrin and various related isostructural biomimetics. Thus, only in the uteroferrin-catalyzed reaction are the precise details of the catalytic mechanism sensitive to the metal ion composition, illustrating the significance of the dynamic ligand environment in the protein active site for the optimization of the catalytic efficiency.

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Acknowledgments

This work was funded by a grant from the Australian Research Council (DP0558652) and CNPq, FAPESC from Brazil. X-ray absorption data collection was performed at the Australian National Beamline Facility (ANBF), Tsukuba, Japan, with support from the Australian Synchrotron Research Program, funded by the Commonwealth of Australia under the Major National Research Facilities Program. We also thank G. Foran for help in data collection. The guidance of Paul Bernhardt in electrochemical measurements with Uf and the assistance of Keith Murray (Monash University, VIC, Australia) with collection of the susceptibility data are kindly acknowledged.

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Authors and Affiliations

  1. School of Molecular and Microbial Sciences, The University of Queensland, St Lucia, QLD, 4072, Australia

    Gerhard Schenk, Andrew K. Dick, Paul Herrald, Mark J. Riley & Lawrence R. Gahan

  2. Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis, SC, 88040-900, Brazil

    Rosely A. Peralta, Suzana Cimara Batista, Adailton J. Bortoluzzi, Bruno Szpoganicz & Ademir Neves

  3. Centre for Magnetic Resonance, The University of Queensland, St Lucia, QLD, 4072, Australia

    Graeme R. Hanson

  4. Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, 59717-3400, USA

    Robert K. Szilagyi

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  1. Gerhard Schenk
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Correspondence to Gerhard Schenk or Ademir Neves.

Electronic supplementary material

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775_2007_305_MOESM1_ESM.pdf

Supporting information available: magnetochemical data for complexes 1 and 3 (Fig. S1), as well as cyclic voltammograms for all three biomimetics (Fig. S2) are available. Saturation kinetics plots and the pH dependence of the catalytic rates for the biomimetics are shown in Figs. S3 and S4. One table of spectroscopic data is also included (Table S1). (PDF 307 kb)

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Schenk, G., Peralta, R.A., Batista, S.C. et al. Probing the role of the divalent metal ion in uteroferrin using metal ion replacement and a comparison to isostructural biomimetics. J Biol Inorg Chem 13, 139–155 (2008). https://doi.org/10.1007/s00775-007-0305-z

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