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Ligation of the Diiron Site of the Hydroxylase Component of Methane Monooxygenase ANELECTRON NUCLEAR DOUBLE RESONANCE STUDY*

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Ligation of the Diiron Site of the Hydroxylase Component of Methane Monooxygenase ANELECTRON NUCLEAR DOUBLE RESONANCE STUDY* THEJOURNAL OF BIOLOGICAL CHEMISTRY VOl. 267, No 1, Issue of January 5, pp. 261-269,1992 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Ligation of the Diiron Site of the Hydroxylase Component of Methane Monooxygenase ANELECTRON NUCLEAR DOUBLE RESONANCE STUDY* (Received for publication, August 5, 1991) Michael P. HendrichSO, Brian G.FoxSlI, Kristoffer K. AnderssonS, Peter G.Debrunner]], and John D. Lipscomb$** From the $Department of Biochemistry, Medical School, University of Minnesota, Minneapolis, Minnesota 55455 and the 11 Department of Physics, University of Illinois, Urbana, Illinois 61801 Electron nuclear double resonance (ENDOR) spec- addition of methanol may result from binding toa site troscopy is used to probe the coordinationof the mixed away from the diiron cluster or from bulk solvent valence (Fe(II)*Fe(III)) diiron clusterof the methane effects on the protein structure. monooxygenase hydroxylase component("OH-) iso- lated from Methylosinus trichosporium OB3b. EN- DOR resonances are observed along the principal axis directions g, = 1.94 and g, = 1.76 from at least nine Soluble methane monooxygenase (EC 1.14.13.25, "0)' different protons and two differentnitrogens. The ni- isolated from Methylosinus trichosporium OB3b is composed trogens are stronglycoupled and appear tobe directly of three protein components(1): a 40-kDa reductase contain- coordinated to the cluster irons. The ratio of their ing FAD and a [2Fe-2S] cluster, a 16-kDa cofactorless com- superhyperfine couplingconstants is roughly 4:7, ponent B, anda 245-kDa hydroxylase with an ((~py)~subunit which equals the ratio of the spin expectation values structure containing two p-(H/R)-oxo-bridged dinuclear iron of the Fe(I1) and Fe(II1) in the ground state and suggestsclusters.' The function of the enzyme in vivo is to catalyze that atleast one nitrogenis coordinated to each ironof the 0; and NADH-dependent oxidationof methane to meth- the mixed valence cluster. Moreover, the superhyper- anol. This is the first step in the catalytic pathway utilized fine and quadrupole coupling constants assigned to bythe methanotrophic bacteria toprovide carbon and energy for Fe(II1) site (AN= 13.6 MHz, PN = 0.7 MHz) are com- growth (4, 5). MMO also catalyzes the adventitiousoxidation parable with those observed for semimethemerythrin of many other saturated and unsaturated, linear, branched, sulfide (AN= 12.1 MHz, PN = 0.7 MHz), for which the and cyclic hydrocarbons (6). nitrogen ligands are histidines. At least three of the Mossbauer and EPR studiesof the hydroxylase component coupled protons exchange slowly when "OH- is in- (MMOH) have characterized some of the electronic properties cubated in D20, and2H ENDOR resonances are subse- of three oxidation states of the diiron site (3, 7). The fully quently observed. These observationsare also consist- oxidized diferric state ("OH') is diamagnetic at low tem- ent with histidine ligation of the iron cluster. On ad- perature and does not give an EPR signal. The one electron dition of the inhibitor dimethyl sulfoxide (Me2SO)to reduced form ("OH-) gives an EPR spectrum inwhich the "OH- the EPR spectrum sharpens and shifts dra- g values are all below 2 (gl= 1.94, g, = 1.86, g3 = 1.76). Such matically. Only one set of 14N ENDOR resonances is spectra are characteristicof Fe(I1).Fe(II1) clusters in proteins observed with frequencies equal to those assigned to and model complexes when the irons experience an antifer- the Fe(II1)-histidineresonances of uncomplexed romagnetic exchange interaction He, = -2JS1.S2 (8, 9); for "OH- suggesting that the nitrogen coordination to "OH- the exchange coupling is J = -30 cm" (10). The the Fe(I1) site is altered orpossibly lost in the presence fully reduced diferrous state ("OH2-) gives rise toan of Me2S0. 2H ENDOR resonances are observed in the integer-spin EPR signal at g = 16 resultingfrom a ferromag- presence of de-Me2S0indicating that the inhibitor netic coupling (J 0) of the irons (7). Me2S0 binds near or possibly to the diiron cluster. In > contrast, no 2H ENDORresonances are observed from MMOH has been identified as the site of the oxygen acti- &-methanol upon additionto "OH-. Thus,the vationand insertion reaction by two experimentalap- changes observedin the EPR spectrumof "OH- upon proaches. First, "OHZ- produced by chemical or electro- chemical reduction in the absence of the other two MMO * This work was supported by Grants GM40466 (to J. D. L.), and components can cycle to "OHo with formation of hydrox- GM16406 (to P. G. D.) from the National Institute of General Medical ylated product when exposed to substrate andO2 (1).Second, Sciences, by a grant from the Center for Biological Process Technol- ogy of the University of Minnesota (to J. D. L.), and by a contract The abbreviations used are: MMO, methane monooxygenase; from Amoco Biotechnology Corporation (to J. D. L.). The costs of MMOH, hydroxylase component of MMO; MMOH', Fe(II1) .Fe(III) publication of this article were defrayed in part by the payment of state of "OH; "OH-, Fe(I1) .Fe(III) stateof "OH; "OH2-, page charges. This article must therefore be hereby marked "adver- Fe(I1) .Fe(II) state of MMOH; Me'SO, dimethyl sulfoxide; EXAFS, tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate extended x-ray absorption fine structure; ENDOR, electron nuclear this fact. double resonance; rf, radio frequency; MOPS, 4-morpholinepropane- I Recipient of National Institute of Health Postdoctoral Fellowship sulfonic acid; MES, 2-(N-morpholino)ethanesulfonicacid. GM12996. 'The chemical state of the bridging oxygen in the hydroxylase (I Recipient of a University of Minnesota Doctoral Dissertation cluster has not been unambiguously established although a proton- Fellowship in partial support of this work. Present address: Dept. of ated or otherwise substituted oxygen is suggested by EXAFS (2) and Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213. Mossbauer (3) studies. We use the general term p-(H/R)-oxo- to ** To whom all correspondence should be addressed. include oxo-, hydroxo-, and R-oxo-ligands. 261 262 ENDOR of MethaneMonooxygenase Hydroxylase Component "OHo alone can catalytically yield product when exposed Samples of "OHo were exchanged into DzO at 4 'C by passage to a substrate and hydrogenperoxide independent of the through anaerobic Sepharose G-25column (12 X 60 mm) equilibrated presence of O2 (11). in 100 mM MOPS buffer, pD 7.0 (as measured by a pH electrode at 4 "C). After exchange, the samples were concentrated by centrifuga- Oxo-bridged diiron clusters are also observed in a variety tion witha centricon YM-30 concentrator (Amicon) to a protein of other proteins (12-14) including hemerythrin, ribonucleo- concentration of nominally 1mM. The total timeallowed for exchange tide reductase, purple acid phosphatases,and uteroferrin. with D20 was at least 8 h. The catalytic activity of MMOH was However, MMOH is the only protein for which the p-(H/R)- unaffected by exchange into D20. For samples containing methanol oxo-bridged diiron cluster is known to catalyzeoxygenase or dl-methanol, thesecompounds were added from1 M stock solutions reactions. For this reason, it is of interest to determine the to anaerobic "OH- samples. structural and electronic properties which make the diiron The sulfide-bridged derivative of semimethemerythrin (19,20)was generouslyprovided by Dr. Donald M. Kurtz,Jr., University of site of MMOH unique. Thus far, no determinationof any of Georgia. the ironligands has been reported,although the EXAFS EPR andENDOR Instrumentation-ENDOR spectra were re- spectra (2) are suggestive of an oxygen or nitrogen environ- corded on a Bruker 200D EPR/ENDOR spectrometer with the mi- ment and the lack of a visible optical spectrum from the crowave bridge tuned to detect the absorption componentof the EPR cluster (1) suggests the absence of ligands capable of charge resonance. The ENDOR rf components consist of a Wavetek fre- transfer interactions such ascysteine and tyrosine. quency synthesizer and a 300W EN1 broad band rf amplifier. The The interaction of substrates with the diiron cluster of microwave/rf probe consistsof a Bruker cylindrical TMo11 cavity and a rf helix (Q = 500 with helix). The rf helix has 18 turns of copper MMOH also remains uncharacterized. Addition of alternate ribbon wrapped around a quartz cryostat finger that is part of an substrates, products, and inhibitors (5, 15, 16) to "OH- Oxford ESR 9 liquid helium cryostat. The rf circuit was connected to have been shown to perturb the EPR spectrum indicating a a 50 ohm load. ENDOR spectra were observed by fixing the magnetic change in the environmentof the cluster.However, it has not field at an EPR resonance and applying partially saturating micro- been previously demonstrated whether the perturbation re- wave power while sweeping the NMR transitionwith a rf source. To sults from the ligation of the small molecules to the cluster, enhance the signal-to-noiseratio, the rf source was frequency modu- binding to the protein near the cluster, or to bulk solvent lated at 12.5 kHz; consequently, the spectral output appears as the effects on the protein structure. derivative of the absorption with respect to frequency. The spectra were digitally recorded on a Bruker Aspect 2000 computer. In this studynew insight into theligation and structure of EPR spectra were recorded on a Varian E109 spectrometer the diiron cluster in "OH- is gained from ENDOR spec- equipped with an Oxford ESR 910 liquid helium cryostat. The mag- troscopy. Moreover, an assessment of the interaction of in- netic field for the EPR and ENDOR measurements was calibrated hibitory molecules is provided. Since few ENDOR studies of with a NMR gaussmeter and themicrowave and rf frequencies were oxo-bridged diiron proteins exist (17), the present study pro- measured with a frequency counter. The analyses (simulation, inte- vides a basis for the interpretation of ENDOR spectra from gration, peak assignment, etc.) of the digitized (1000 points) EPR this important proteinclass.
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