Type 2 Cu2+ in pMMO from Methylomicrobium album BG8

ABSTRACT EPR spectra were obtained for the type 2 Cu^sup 2+^ site in particulate methane monooxygenase (pMMO) from Methylomicrobium album BG8 grown on K^sup 15^N0^sub 3^ and ^sup 63^Cu(N0^sub 3^)^sub 2^. The concentration of the type 2 Cu2+ signal was -200 (mu)M per 25 mg/ml protein in packed cells and membrane fractions, a concentration that is consistent with its attribution to pMMO, and the EPR parameters were consistent with electron paramagnetic resonance (EPR) parameters previously assigned to pMMO. The superhyperfine structure due to nitrogen is better resolved because I = 1/2 for '5N whereas I = 1 for ^sup 14^N and A(^sup 15^N)/A(^sup 14^N) = 1.4. Under these conditions, superhyperfine structure is resolved in the g^sub ||^ region of the X-band spectrum. At low microwave frequency (S-band) the resolution of the nitrogen superhyperfine structure improves. Signals are attributed to type 2 Cu^sup 2+^ in which cupric ion is bound to four (less likely three) nitrogen donor atoms.

INTRODUCTION

Methane monooxygenases (MMOs) are enzymes that convert methane to methanol (Hanson et al., 1991; Hanson and Hanson, 1996; Anthony, 1986).

Distinct soluble (sMMO) and particulate (pMMO) enzymes occur. Much is known about the oxo-bridged dinuclear iron center in sMMO (Rosenzweig et al., 1993; Lipscomb, 1994), but much less is known about the copper center(s) in pMMO. Whereas pMMO is present in all known methanotrophic bacteria, sMMO is limited to some strains and is made only under conditions of copper limitation (Prior and Dalton, 1985). The pMMO from Methylococcus capsulatus (Bath) consists of three subunits of 47, 27, and 25 kDa. Electron spin paramagnetic resonance (EPR)-detectable Cu2+ from pMMO in cells has been correlated to activity (Nguyen et al., 1994, 1996; Chan et al., 1993). Chan, Lidstrom, and co-workers have proposed a mechanism for dioxygen reduction and methane activation by a trinuclear copper cluster (Chan et al., 1993). Recently, mechanistic hypotheses (Elliott et al., 1997) for dioxygen adducts based on a dicopper-oxo core, as described in model complexes (Halfen et al., 1996; Mahapatra et al., 1996), included three schemes involving direct insertion of an activated oxygen atom into the pro-(R) C-H bond or a concerted pairwise process or hydrogen atom abstraction followed by attack of an oxygen-based radical. Regardless of the mechanism, alkane hydroxylation proceeds favoring attack at the C-2 position (Elliott et al., 1997). DiSpirito and co-workers have presented an alternative model in which the catalytic site involves both iron and copper, although they reserve the option of a single ferrous iron center or an iron-iron center (Zahn and DiSpirito, 1996).

The source of pMMO for many of the studies is M. capsulatus (Bath), which is capable of making both pMMO and sMMO. In this work, the source of pMMO is Methylomicrobium album BG8, in which only copper-loaded pMMO is present. Analysis of pMMO from an organism incapable of making sMMO is advantageous because it has been suggested that under some circumstances sMMO can co-purify with pMMO (Nguyen et al., 1998). It is possible that the differences reported in studies of pMMO from M. capsulatus (Bath) are attributable to sMMO. In our previous EPR study of signals from M. album BG8 (Yuan et al., 1997), we concluded that the cupric EPR signal obtained as isolated or upon reduction of pMMO is not a signal from a mixed valence delocalized copper trimer as previously described by others (Nguyen et al., 1994). Either three or four nitrogen donor atoms contribute to the nitrogen superhyperfine pattern on the MI = -1/2 line in the gl region, but it was too difficult to distinguish between a seven-line pattern with relative intensities of 1:3:6:7:6:3:1 for three approximately equivalent nitrogens and a nine-line pattern with relative intensities of 1:4:10:16:19:16:10:4:1 for four equivalent nitrogens, especially when the outer lines with intensity of 1 are buried in the noise. The data reported here are consistent with four nitrogens bound in a square planar configuration from analysis of whole cells grown on a 15N isotope. Whole cells are used for this analysis to preserve the environment around the copper.

MATERIALS AND METHODS

Growth of bacterium

M. album BG8 was grown in the batch culture in nitrate mineral salts (NMS) medium (Whittenbury et al., 1970) containing 5 AM cupric sulfate. Cultures were incubated at 30C with shaking at 150 rpm in a 50% methane, 50% air (v/v) atmosphere. After growth for 2 days, cells were harvested by centrifugation at 8000 rpm for 30 min at 4oC. The cell pellets were washed twice in ice-cold 20 mM phosphate, 5 mM magnesium chloride buffer (pH 7.0) and collected at 10,000 rpm for 10 min. For isotope studies, cells were grown in the same medium by the same process except potassium nitrate was replaced by 5N-potassium nitrate (Aldrich, Milwaukee, WI) and cupric sulfate replaced by 63-cupric nitrate (Cambridge Isotope Laboratories, Andover, MA).

Enzyme activity

MMO activity was detected by propylene oxidation by whole cells. Cells grown with sufficient copper to saturate the growth requirements in the medium (Collins et al., 1991; Brantner et al., 1997) had an optimal MMO activity under our conditions of 21 nmol/min/mg protein.

Peptide synthesis

A 20-amino-acid peptide, HGEKSQAAFMRMRTIHWYDL, was synthesized by solid-state methods on an automated Milligen Biosearch peptide synthesizer (model 9050) at the Protein/Nucleic Acid Shared Facility at the Medical College of Wisconsin. After synthesis, the peptide was reduced by dithiothreitol and purified by high-pressure liquid chromatography. The purified peptide was then stored in sealed test tubes purged with argon.

A stock solution was prepared by dissolving 1.2 mg of peptide in 150 (mu)l of 0.1 of 0. 1% trifluoroacetic acid (TFA) in double-distilled water. A series of samples was prepared by adding 63Cu(No3)2 to the peptide according to the molar ratio of copper to peptide. Samples with ratios of Cu2+ to peptide of 1:1,1:2,1:3, and 1:4 were diluted by HEPES buffer (pH 7.2), and the pH was adjusted before freezing the sample in liquid nitrogen.

EPR measurements

All X-band spectra were recorded on a Varian E109 Century Series spectrometer with a Varian TEI 02 cavity (Varian, Palo Alto, CA). Samples were placed in a finger dewar filled with liquid nitrogen. The concentration of Cu2+ in the sample was calculated by comparing the double integral of the spectrum with the double integral of the spectrum from a 1.0 mM cupric perchlorate solution. S-band (3.4 GHz) spectra were obtained from a spectrometer with a loop-gap resonator cavity and a low-frequency microwave bridge built at the National Biomedical ESR Center, Medical College of Wisconsin. S-band samples were cooled by nitrogen passed through an exchange coil immersed in liquid nitrogen. Microwave frequencies were measured with a frequency counter (EIP model 548), and the field was calibrated with a magnetometer (Rawson-Lush Instrument Co., Acton, MA). Spectra were simulated using a program from Lynn Belford (University of Illinois, Urbana, IL) (Nigles, 1979; Maurice, 1980).

RESULTS AND DISCUSSION

We thank Liane M. Mende-Mueller, Protein/Nucleic Acid Shared Facility, and Emily Marks, an intern from Alverno College, for their contribution with respect to the data from the protein fragment. This work was supported by grant RR01008 from the National Institutes of Health and by grant MCB-9118653 from the National Science Foundation and is publication 417 from the Center for Great Lakes Studies.

[Reference]

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[Author Affiliation]

Hua Yuan,* Mary Lynne Perille Collins,# and William E. Antholine*

*Biophysics Institute, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and #Department of Biological Sciences and Great Lakes Water Institute, The University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201 USA

[Author Affiliation]

Address reprint requests to Dr. William E. Antholine, Biophysics Research Institute, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. Tel.: 414456-4032; Fax: 414-456-6512; E-mail: wantholi@mcw.edu.

H. Yuan's current address: The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037.