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Licensed Unlicensed Requires Authentication Published by De Gruyter June 12, 2018

Spectroscopic characterization of the Co-substituted C-terminal domain of rubredoxin-2

  • Lisa M. Galle , George E. Cutsail III , Volker Nischwitz , Serena DeBeer and Ingrid Span ORCID logo EMAIL logo
From the journal Biological Chemistry


Pseudomonas putida rubredoxin-2 (Rxn2) is an essential member of the alkane hydroxylation pathway and transfers electrons from a reductase to the membrane-bound hydroxylase. The regioselective hydroxylation of linear alkanes is a challenging chemical transformation of great interest for the chemical industry. Herein, we report the preparation and spectroscopic characterization of cobalt-substituted P. putida Rxn2 and a truncated version of the protein consisting of the C-terminal domain of the protein. Our spectroscopic data on the Co-substituted C-terminal domain supports a high-spin Co(II) with a distorted tetrahedral coordination environment. Investigation of the two-domain protein Rxn2 provides insights into the metal-binding properties of the N-terminal domain, the role of which is not well understood so far. Circular dichroism, electron paramagnetic resonance and X-ray absorption spectroscopies support an alternative Co-binding site within the N-terminal domain, which appears to not be relevant in nature. We have shown that chemical reconstitution in the presence of Co leads to incorporation of Co(II) into the active site of the C-terminal domain, but not the N-terminal domain of Rxn2 indicating distinct roles for the two rubredoxin domains.


This work was supported by the Fonds der Chemischen Industrie, Liebig Fellowship to I.S. and a PhD fellowship to L.M.G. The authors also thank the Alexander von Humboldt Foundation (to G.E.C) and the Max-Planck-Gesellschaft (to S.D.). The European Synchrotron Radiation Facility is acknowledged for providing beamtime and for technical support from Lucia Amidani at beam line ID26. We gratefully acknowledge the performance of ICP-MS measurements by Ulrike Seeling and Astrid Küppers, ESI-MS measurements by Sabine Metzger at Leibniz Research Institute for Environmental Medicine, purification using HPLC by Nadine Rösener and Lothar Gremer, and IR spectroscopic measurements by Claudia Hoppen and Georg Groth. We are also grateful to Manuel Etzkorn and Peter Bayer for providing plasmids, and Dieter Willbold for the laboratory facilities. We acknowledge networking support to L.M.G. and I.S. from the COST action FeSBioNet (contract CA15133).

  1. Conflict of interest statement: The authors declare that they have no conflict of interest regarding the contents of this article.


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Received: 2018-01-31
Accepted: 2018-04-25
Published Online: 2018-06-12
Published in Print: 2018-06-27

©2018 Walter de Gruyter GmbH, Berlin/Boston

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