Jump to ContentJump to Main Navigation
Show Summary Details
More options …

Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred

IMPACT FACTOR 2018: 3.014
5-year IMPACT FACTOR: 3.162

CiteScore 2018: 3.09

SCImago Journal Rank (SJR) 2018: 1.482
Source Normalized Impact per Paper (SNIP) 2018: 0.820

See all formats and pricing
More options …
Volume 399, Issue 7


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

Lisa M. Galle
  • Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ George E. Cutsail III / Volker Nischwitz
  • Central Institute for Engineering, Electronics and Analytics (ZEA-3), Forschungszentrum Jülich, D-52425 Jülich, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Serena DeBeer / Ingrid SpanORCID iD: http://orcid.org/0000-0002-2892-4825
Published Online: 2018-06-12 | DOI: https://doi.org/10.1515/hsz-2018-0142


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 article offers supplementary material which is provided at the end of the article.

Keywords: AlkG; iron-sulfur protein; metal substitution; Pseudomonas putida GPo1; rubredoxin


  • Anglin, J.R. and Davison, A. (1975). Iron(II) and cobalt(II) complexes of Boc-(Gly-L-Cys-Gly)4-NH2 as analogs for the active site of the iron-sulfur protein rubredoxin. Inorg. Chem. 14, 234–237.CrossrefGoogle Scholar

  • Bencini, A., Bertini, I., Canti, G., Gatteschi, D., and Luchinat, C. (1981). The epr spectra of the inhibitor derivatives of cobalt carbonic anhydrase. J. Inorg. Biochem. 14, 81–93.PubMedCrossrefGoogle Scholar

  • Bordeaux, M., Galarneau, A., and Drone, J. (2012). Catalytic, mild, and selective oxyfunctionalization of linear alkanes: current challenges. Angew. Chem. Int. Ed. 51, 10712–10723.CrossrefWeb of ScienceGoogle Scholar

  • Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.CrossrefPubMedGoogle Scholar

  • Castillo, R.G., Banerjee, R., Allpress, C.J., Rohde, G.T., Bill, E., Que, L., Lipscomb, J.D., and DeBeer, S. (2017). High-energy-resolution fluorescence-detected X-ray absorption of the Q intermediate of soluble methane monooxygenase. J. Am. Chem. Soc. 139, 18024–18033.PubMedCrossrefWeb of ScienceGoogle Scholar

  • Curdel, A. and Iwatsubo, M. (1968). Biosynthetic incorporation of cobalt into yeast alcohol dehydrogenase. FEBS Lett. 1, 133–136.PubMedCrossrefGoogle Scholar

  • Dauter, Z., Wilson, K.S., Siekert, L.C., Moulist, J.-M., and Meyer, J. (1996). Zinc-and iron-rubredoxins from Clostridium pasteurianum at atomic resolution: a high-precision model of a ZnS4 coordination unit in a protein. Biochemistry 93, 8836–8840.Google Scholar

  • Drum, D.E. and Vallee, B.L. (1970). Optical properties of catalytically active cobalt and cadmium liver alcohol dehydrogenases. Biochem. Biophys. Res. Commun. 41, 33–39.CrossrefPubMedGoogle Scholar

  • Edelhoch, H. (1967). Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6, 1948–1954.CrossrefPubMedGoogle Scholar

  • Fukui, K., Ohya-Nishiguchi, H., and Hirota, N. (1991). ESR and magnetic susceptibility studies on high-spin tetrahedral cobalt(II)–thiolate complexes: an approach to rubredoxin-type active sites. Bull. Chem. Soc. Jpn. 64, 1205–1212.CrossrefGoogle Scholar

  • Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D., and Bairoch, A. (2005). Protein identification and analysis tools on the ExPASy server. In: The Proteomics Protocols Handbook, John M. Walker, ed. (Totowa, NJ, USA: Humana Press), pp. 571–607.Google Scholar

  • Gavel, O.Y., Bursakov, S.A., Calvete, J.J., George, G.N., Moura, J.J.G., and Moura, I. (1998). ATP sulfurylases from sulfate-reducing bacteria of the genus Desulfovibrio. A novel metalloprotein containing cobalt and zinc. Biochemistry 37, 16225–16232.PubMedCrossrefGoogle Scholar

  • Getz, E.B., Xiao, M., Chakrabarty, T., Cooke, R., and Selvin, P.R.A. (1999). A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry. Anal. Biochem. 273, 73–80.PubMedCrossrefGoogle Scholar

  • Gill, S.C. and von Hippel, P.H. (1989). Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319–326.PubMedCrossrefGoogle Scholar

  • Good, M. and Vasak, M. (1986). Spectroscopic properties of the cobalt(II)-substituted α-fragment of rabbit liver metallothionein. Biochemistry 25, 3328–3334.PubMedCrossrefGoogle Scholar

  • Kok, M., Oldenhuis, M., van der Linden, M.P.G., Meulenberg, C.H.C., Kingma, J., and Withold, B. (1989). The Pseudomonas oleovorans alkBAC operon encodes two structurally related rubredoxins and an aldehyde dehydrogenase. J. Biol. Chem. 264, 5442–5451.PubMedGoogle Scholar

  • Lee, H.J., Lian, L.Y., and Scrutton, N.S. (1997). Recombinant two-iron rubredoxin of Pseudomonas oleovorans: overexpression, purification and characterization by optical, CD and 113Cd NMR spectroscopies. Biochem. J. 328, 131–136.CrossrefPubMedGoogle Scholar

  • Lode, E.T. and Coon, M.J. (1971). Enzymatic omega-oxidation. V. Forms of Pseudomonas oleovorans A containing one or two iron atoms: structure and function in omega-hydroxylation. J. Biol. Chem. 246, 791–802.PubMedGoogle Scholar

  • Lovenberg, W. and Sobel, B.E. (1965). Rubredoxin: a new electron transfer protein from Clostridium pasteurianum. Proc. Natl. Acad. Sci. USA 54, 193–199.CrossrefGoogle Scholar

  • Maher, M., Cross, M., Wilce, M.C.J., Guss, J.M., and Wedd, A.G. (2004). Metal-substituted derivatives of the rubredoxin from Clostridium pasteurianum. Acta Crystallogr. D Biol. Crystallogr. 60, 298–303.CrossrefPubMedGoogle Scholar

  • Majtan, T., Freeman, K.M., Smith, A.T., Burstyn, J.N., and Kraus, J.P. (2011). Purification and characterization of cystathionine β-synthase bearing a cobalt protoporphyrin. Arch. Biochem. Biophys. 508, 25–30.PubMedCrossrefWeb of ScienceGoogle Scholar

  • Makinen, M.W., Kuo, L.C., Yim, M.B., Wells, G.B., Fukuyama, J.M., and Kim, J.E. (1985). Ground term splitting of high-spin cobalt(2+) ion as a probe of coordination structure. 1. Dependence of the splitting on coordination geometry. J. Am. Chem. Soc. 107, 5245–5255.CrossrefGoogle Scholar

  • Maret, W. and Vallee, B.L. (1993). Cobalt as probe and label of proteins. Methods Enzymol. 226, 52–71.CrossrefPubMedGoogle Scholar

  • May, S.W. and Kuo, J.Y. (1978). Preparation and properties of cobalt(II) rubredoxin. Biochemistry 17, 3333–3338.PubMedCrossrefGoogle Scholar

  • McMillin, D.R., Holwerda, R.A., and Gray, H.B. (1974). Preparation and spectroscopic studies of cobalt(II)-stellacyanin. Proc. Natl. Acad. Sci. USA 71, 1339–1341.CrossrefGoogle Scholar

  • Moura, I., Teixeira, M., Moura, J.J.G., and LeGall, J. (1991). Spectroscopic studies of cobalt and nickel substituted rubredoxin and desulforedoxin. J. Inorg. Biochem. 44, 127–139.CrossrefPubMedGoogle Scholar

  • Perry, A., Lian, L.Y., and Scrutton, N.S. (2001). Two-iron rubredoxin of Pseudomonas oleovorans: production, stability and characterization of the individual iron-binding domains by optical, CD and NMR spectroscopies. Biochem. J. 354, 89–98.CrossrefPubMedGoogle Scholar

  • Perry, A., Tambyrajah, W., Grossmann, J.G., Lian, L.Y., and Scrutton, N.S. (2004). Solution structure of the two-iron rubredoxin of Pseudomonas oleovorans determined by NMR spectroscopy and solution X-ray scattering and interactions with rubredoxin reductase. Biochemistry 43, 3167–3182.CrossrefPubMedGoogle Scholar

  • Ravel, B. and Newville, M. (2005). ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT. J. Synchrotron Radiat. 12, 537–541.PubMedCrossrefGoogle Scholar

  • Shimizu, T., Mims, W.B., Davis, J.L., and Peisach, J. (1983). Studies of the coordination of rare earth and transition metal nucleotide complexes by an electron spin echo method. Biochim. Biophys. Acta 757, 29–39.CrossrefGoogle Scholar

  • Stoll, S. and Schweiger, A. (2006). EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J. Magn. Reson. 178, 42–55.CrossrefPubMedGoogle Scholar

  • Strug, I., Utzat, C., Cappione, A., Gutierrez, S., Amara, R., Lento, J., Capito, F., Skudas, R., Chernokalskaya, E., and Nadler, T. (2014). Development of a univariate membrane-based mid-infrared method for protein quantitation and total lipid content analysis of biological samples. J. Anal. Methods Chem. 2014, 1–12.CrossrefWeb of ScienceGoogle Scholar

  • Sugiura, Y. (1978). Electronic properties of sulfhydryl- and imidazole-containing peptide-cobalt(II) complexes: their relationship to cobalt(II)-substituted “blue” copper proteins. Bioinorg. Chem. 8, 453–460.CrossrefPubMedGoogle Scholar

  • Thomas, J.M., Raja, R., Sankar, G., and Bell, R.G. (2001). Molecular sieve catalysts for the regioselective and shape-selective oxyfunctionalization of alkanes in air. Acc. Chem. Res. 34, 191–200.CrossrefPubMedGoogle Scholar

  • Tsai, Y.-F., Luo, W.-I., Chang, J.-L., Chang, C.-W., Chuang, H.-C., Ramu, R., Wei, G.-T., Zen, J.-M., and Yu, S.S.-F. (2017). Electrochemical hydroxylation of C3–C12 n-alkanes by recombinant alkane hydroxylase (AlkB) and rubredoxin-2 (AlkG) from Pseudomonas putida GPo1. Sci. Rep. 7, 1–13.Google Scholar

  • van Beilen, J.B., Neuenschwander, M., Smits, T.H.M., Roth, C., Balada, S.B., and Witholt B. (2002). Rubredoxins involved in alkane oxidation. J. Bacteriol. 184, 1722–1732.PubMedCrossrefGoogle Scholar

  • Westre, T.E., Kennepohl, P., DeWitt, J.G., Hedman, B., Hodgson, K.O., and Solomon, E.I. (1997). A multiplet analysis of Fe K-edge 1s → 3d pre-edge features of iron complexes. J. Am. Chem. Soc. 119, 6297–6314.CrossrefGoogle Scholar

  • Zielazinski, E.L., Cutsail, G.E., Hoffman, B.M., Stemmler, T.L., and Rosenzweig, A. (2012). Characterization of a cobalt-specific P1B-ATPase. Biochemistry 51, 7891–7900.CrossrefGoogle Scholar

About the article

Received: 2018-01-31

Accepted: 2018-04-25

Published Online: 2018-06-12

Published in Print: 2018-06-27

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

Citation Information: Biological Chemistry, Volume 399, Issue 7, Pages 787–798, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/hsz-2018-0142.

Export Citation

©2018 Walter de Gruyter GmbH, Berlin/Boston.Get Permission

Supplementary Article Materials

Comments (0)

Please log in or register to comment.
Log in