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Laue crystal structure of Shewanella oneidensis cytochrome c nitrite reductase from a high-yield expression system

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Abstract

The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein “small tetraheme c” replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5–1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 °C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-Å-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein).

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Abbreviations

ccNiR:

Cytochrome c nitrite reductase

CCP4:

Collaborative Computational Project Number 4

HAO:

Hydroxylamine oxidoreductase

HEPES:

N-(2-Hydroxyethyl)piperazine-N′-ethanesulfonic acid

MV:

Methyl viologen

MVred :

Methyl viologen monocation radical

OTTLE:

Optically transparent thin-layer electrode

PFV:

Protein film voltammetry

SHE:

Standard hydrogen electrode

STC:

Small tetraheme c

SVD:

Singular value decomposition

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Acknowledgments

M.S. and A.A.P. are supported by grants NSF-0843459, NSF-1121770, and UWM RGI 101X157. M.S. is supported by NSF CAREER grant 0952643. E.T.J. is supported by an NIH-NIGMS National Research Service Award (NRSA) fellowship (1F31GM099416-01). B.S. and T.G. gratefully acknowledge support from University of Wisconsin-Milwaukee’s Office of Undergraduate Research. Use of the BioCARS Sector 14 was supported by grants from the National Center for Research Resources (5P41RR007707) and the National Institute of General Medical Sciences (8P41GM103543) from the National Institutes of Health. The time-resolved setup at Sector 14 was funded in part through a collaboration with Philip Anfinrud (NIH/NIDDK). Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under contract no. DE-AC02-06CH11357. The authors gratefully acknowledge Nick Silvaggi, Chuck Wimpee, Graham Moran, and Daad Safarini of the University of Wisconsin-Milwauke for helpful discussions, Victor Trussell of Rufus King High School for help in stability studies of ccNiR, Nick Silvaggi for the use of his high-performance chromatography apparatus, and the Jianhua Fu research group (Medical College of Wisconsin) for the use of their automated crystallization machine in the preliminary crystallization screens.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, 3210 N. Cramer St, Milwaukee, WI, 53211, USA

    Matthew Youngblut, Bilal Sayyed, Tyler Goelzer & A. Andrew Pacheco

  2. Department of Physics, University of Wisconsin-Milwaukee, 1900 E. Kenwood Blvd, Milwaukee, WI, 53211, USA

    Marius Schmidt

  3. Department of Chemistry, Boston University, 590 Commonwealth Ave., Boston, MA, 02215, USA

    Evan T. Judd & Sean J. Elliott

  4. Center for Advanced Radiation Sources, The University of Chicago, 9700 S. Cass Ave, Argonne, IL, 60439, USA

    Vukica Srajer

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  1. Matthew Youngblut
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Correspondence to Sean J. Elliott, Marius Schmidt or A. Andrew Pacheco.

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Youngblut, M., Judd, E.T., Srajer, V. et al. Laue crystal structure of Shewanella oneidensis cytochrome c nitrite reductase from a high-yield expression system. J Biol Inorg Chem 17, 647–662 (2012). https://doi.org/10.1007/s00775-012-0885-0

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