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 termp-(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 substrateand 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. andENDOR spectra were performed onan IBM-PC compatible computer using software written by the authors. EXPERIMENTALPROCEDURES RESULTS Protein Purification-The growth of M. trichosporium OB3b and the purification and characterization of MMOH of the soluble meth- EPR Spectroscopy of MMOH--The low temperature EPR ane monooxygenase were as previously described (1, 18). spectrum of"OH-, shown in Fig. lA, is composed of a Chemicals-MOPS, MES, sodium ascorbate, and phenazine meth- majority species (85%, g, = 1.94, gz = 1.86, g3 = 1.76) and a osulfate were obtained fromSigma. D20,ds-MezSO, and d,-methanol were all of 99% or greater purity and were obtained from Aldrich. Water was deionized and glass distilled. All other chemicals were reagent grade or better andused without purification. General Procedures-Enzyme activity wasroutinely assayedas previouslydescribed (1, 18) by monitoring the rate of 02 uptake during furan oxidation using a Clarke type oxygen electrode at room A temperature. The MMOH utilized had a specific activity of between 800 and 1400 nmol/min/mg. Similar values were obtained by meas- uring the rate of propene oxidation to propene oxide with gas chro- B matography at 30 "C. Proteinconcentrations were determinedas previously described (1).Anaerobic conditions were established by repeated cycling between vacuum and argon atmospheres. Traces of 0, were removed from the argon by passage over a BASF copper catalyst at 150 "C. Anaerobic sample transfers were made using gas 1.80 tight Hamilton syringes. I Sample Preparations-ENDORand EPR samples of "OH- C 1/ (nominally 1 mM in a total volume of 180-250 ~l)were prepared under anaerobic conditions in Teflon-sealed reaction vials. One of two reduction procedures were used: 1) addition of 1 equivalent of sodium dithionite (2 reducing equivalents assuming the presence of two diiron clusters in MMOH) in 100 mM MOPS buffer, pH 7.0 (or I, Ill MES buffer pH, 6.0). plus 0.5 mM phenazine methosulfate followed 290 322 354386418 50 byincubation for5 min at 25 "C; 2) addition of 10 mM sodium WmT) ascorbate to "OHo in the presence of 0.3 M MezSO or d6-Me2S0 in 100 mM MOPS, pH 6.5 or 7.0, plus 0.2 mM phenazine methosulfate FIG. 1. X-band EPR spectra of "OH-. Nominally 1 mM followed by incubation for5 min at 25 "C. After addition of the hydroxylase was partially reduced to maximize the "OH- in: A, appropriate reductant, the sampleswere transferred anaerobically to 100 mM MES buffer, pH 6.0; B, 100 mM MOPS buffer, pH 7.0, in 50 a serum-stoppered EPR tube and then frozen in liquid nitrogen. All mM methanol; and C, 100 mM MOPS buffer, pH 7.0, in 0.3 M Me2S0. buffers contain 4% glycerol. Sodium dithionite was prepared in an- Spin quantitation found mixed valence cluster concentrations of 0.6 aerobic, buffered solutions and quantified before use by titration of f 0.1 mM (uersus a Cu" standard) for these samples. Signal inten- potassium ferricyanide solutions (erlO = 1.03 mM" cm-I). Concen- sitiesare normalized to give equalsecond integrals of the mixed trated solutions of sodium ascorbate were prepared daily in anaerobic, valence species. Instrumental parameters: temperature, 10 K; micro- doubly distilled water. wave power, 0.02 milliwatts at 9.130 GHz; gain, 4000. ENDOR of Methane Monooxygenase HydroxylaseComponent 263
minority species (15%, g = 1.98, 1.87, 1.70); in addition to a IAH/2 f vHI, where AHis the superhyperfinecoupling constant resonance at g = 2.00 which is preparation dependent and has and vH is the proton Larmor frequency (14.90 MHz for 350 been previously assigned to a radical species(1). The spectrum millitesla). The proton ENDOR spectrumof "OH- at gl is of "OH- plus product (50 mM methanol) is shown in Fig. shown in Fig. 2A. All ENDOR spectra presentedin this paper 1B (21). The resonance atg, is sharper, but the resonance at are only observed within the narrow temperaturerange of 7- g3 is broader and has shifted to g, = 1.74. The spectrum of 14 K. For temperatures outside thisrange, unfavorable relax- "OH- plus 0.3 M MezSO is shown in Fig. 1C; all features ation rates quickly diminish the ENDOR effect. The proton are sharper. As with methanol, the addition of MezSO does spectra are plotted as v - vH, so that the AH value for a pair not significantly change the values of gl and g,, but g3 shifts of resonancesis given by twice the absolutevalue of the in this case to 1.80. The sample homogeneity appears to be frequency of a resonance position. At least nineresolved pairs improved upon addition of Me,SO, since the intensityof the of proton resonances are observed as noted with arrows on minority species is significantly reduced. The half-width at the lower frequency half of each pair shown in Fig. 2A. The half-heights of the glfeature for "OH- in water, methanol, AH values for all resonances observed in Fig. 2 are listed in and MezSO are 2.9, 1.8, and 1.3 millitesla, respectively. Sam- Table I. The resonance corresponding to the largest super- ples equivalent to those of Fig. 1, A-C, were prepared instead hyperfine constant, AH = 7.8 MHz, is noted with anasterisk with DzO (A), &-methanol (B),and d6-MezS0(C). Compar- and will be referred to later. The EPR spectrumof "OH- ison of the features at glfor these samples showed no change has more than onespecies, thus proton resonances withweak in linewidth relative to the proticequivalent. intensity (e.g. thepair with IAH/21 = 2.4 MHz) may be Both methanol and Me2S0 were found to function as in- associated with a minority species. The proton spectrum at hibitors of the reconstituted enzyme system. However, the the other EPR extremum,g3, shown in Fig. 2B, contains six inhibition did not conform to a single class in either case, obviating direct determination of K, values. In a standard assay containing 4 mM furan, 50% inhibition was observed on addition of 6 mM methanol or 12 mM Me,SO. Both small molecules can be removed from the enzyme by gel filtration techniques to restore full activity. Moreover, an EPR spec- trum of "OH- like that shown in Fig. lA is observed after the small molecule is removed. The EPR spectra of samples for which thediiron cluster is reduced before addition of > either methanol or MezSO are equivalent to the respective 1 1 11 11 x EPR spectra of samples that were reduced after small mole- U cule addition. Mossbauer spectra of the complexes formed by these molecules with "OHo show that the diiron cluster remainsantiferromagnetically coupled andno measurable amount of mononuclear ferrous or ferric iron is formed when the complexes are made.4 The samples of "OH- giving the EPR spectraof Fig. 1 were used for the ENDOR measurements presented here. Currenttechniques designed to maximize "OH- give IIIIIIIIII -4 -2 0 2 4 roughly equal amountsof all three oxidation states(3). Thus, v - VH (MHz) EPR signals at g = 16 from "OH2- and at g < 2 from "OH- are observed from these samples. ENDOR spectra FIG. 2. Proton ENDOR spectra of "OH-. A, g, = 1.94, pH = were observed for the g < 2 signal; the integer spin g = 16 14.88 MHz; B, g3 = 1.77, pH = 16.29 MHz. The position of the lower frequency resonance of each proton pairis noted with an arrow. The signal has weak intensity at g = 2 (7) and, thus, should not vertical bars indicate an empirical correlation of resonances between affect ENDOR signals from themixed valence form. Although g, and g3. The sample conditions are given in Fig. 1A. The spectra the EPR signalsfrom "OH- represent at best one-thirdof are normalized to the samevertical scale and the off-resonance base the total iron (3), ENDOR spectra were observed from 'H, line has been subtracted. Instrumental parameters: temperature, 10 2H, 14N,and 57Fe (enriched) with an adequatesignal-to-noise K; microwave power, 50 milliwatts at 9.495 GHz; gain, 1.25 X lo6; ratio for protein concentrations of nominally 1 mM. The 57Fe time constant, 0.1 s; 65 scans; rf sweep rate, 0.33 MHz/s; frequency ENDOR results will be presented elsewhere in combination modulation, k25 kHz at 12.5 kHz; rf attenuation, 6 dB. with theassociated Mossbauer spectroscopy. Frozen solution ENDOR spectra are simplest to interpret TABLEI at the extrema, g, or g3, of the EPR spectrum. The ENDOR Proton superhyperfine constants at g, for "OH- spectra at these two g values are composed of NMR reso- Sample conditions are given in Fig. 1. Abbreviations: ex, exchange- nances from a single orientation of the diiron site withrespect able in D20;$, empirical correlation of AHvalues observed at g, and to themagnetic field, thus sharp resonances areobserved. In ~3,A,H, = 0.9 ApH3. Addition Principal this study, we present data taken at thesetwo principal axis A" axis g values. The interpretationof the full angular setof ENDOR data taken at intermediate g values (cf. Ref. 22) requires MHz additional measurements. None g1 7.8,4.8, 3.6$, 3.1, 2.7$,,,1.8$,,, 1.3$,,, 0.9, 0.7$ 'H,'H ENDOR Spectroscopy-The ENDOR spectrum from g3 4.1$, 3.0$.,, 2.0$,1.5$,,, 0.8$, 0.4 'H (I = 1/2) consists of two resonances at frequencies v = Methanol" g, 7, 4.1, 3.5, 2.9.,, 1&, 1.L, 0.7, 0.3
The relative amounts of species are estimated from double inte- MezSO g1 7.9, 6.7, 2.8$, 2.5$, 2.1, 1.4$, 0.8, 0.3 gration of simulated EPR spectrausing the given g values. 23 13.0, 9.1, 4.4, 3.2$, 2.8$, 2.2, 1.6$, 0.8, 0.4 B. G. Fox, E. Munck, K. K. Andersson, M. P. Hendrich, and J. No values at g3 are given due to the broad EPR resonance and D. Lipscomb, unpublished result. concomitant low signal-to-noise ratio of the ENDOR spectrum. 264 ENDOR of Methane Monooxygenme Hydroxylase Component resolved pairs of resonances. It is observed that five pairs of resonances at gl and g3 obey the empirical relation, A2 = 0.9 AS; these are noted in Fig. 2 and Table I. Water Accessibility to theDiiron Cluster-"OHo was incubated in DzO following the protocol given under "Exper- imental Procedures" for a period of 8 h, after which "OH- was prepared. The proton ENDOR spectra of "OH- at gl in HzO (solid line) and DzO (dashed line) are overlaid in Fig. 3A. The difference spectrum, Fig. 3B, indicates that at least three protons are exchangeable. The resonances andAHvalues of these protons are noted with arrows in Fig. 3B and with "ex" in Table I. Roughly half of the intensity of the proton resonances with AH = 2.7 MHz is lost upon sample deutera- tion. The resonances with AH = 7.8 MHz are broader for this sample, thus it is difficult to determine from the data pre- sented in Fig. 3, A and B, if the proton is exchangeable. However, an examination of these resonances from several IIIIIIIII other data setsindicates that thisproton is not exchangeable. 123456 The difference ENDOR spectrum HzO-DzOat g3 (not shown) v (MHz) shows at least two exchangeable protons with AH values of 3.0 and 1.5 and with intensity similar in magnitude to those FIG. 4. Deuterium ENDOR difference spectra at g, of "OH-. "OH- in 100 mM MOPS buffer, pD 7.0, prepared in at g,. Notice that theAH values of these two protons obey the DzO (A),methanol/DZO (B),d4-methanol/Dz0 (C), ds-MezSO/HzO empirical relation A$ = 0.9 AS and the corresponding pro- (D)(pH 7.0). The spectrum of the equivalent samples having no tons atgl are exchangeable. deuterium have been subtracted from each and the resulting spectra The effect of deuterium exchange is also evident from the are normalized to the same vertical scale. The vertical bar is located observation of a 'H ENDOR resonance at gl as shown in Fig. at the deuteriumLarmor frequency, uD = 2.25 MHz. The sample The difference spectrum DzO-HzO shows two features conditions are given in Figs. 3 and 5. Instrumental parameters: 4A. temperature, 10 K; microwave power, 20 milliwatts at GHz; gain, near the deuterium Larmor frequency &' (2.29 MHz for 350 9.38 1 X lo6; time constant, 0.2 s; 15 scans; rf sweep rate, 0.2 MHz/s; millitesla). The 'H signal is also observed at g3 (not shown), frequency modulation, f100 kHz at 12.5 kHz; rf attenuation, 6 dB. shifted to slightly higher frequency in accordance with the increase in magnetic field at g3. Due to the anomalous line- buffered HzO or DzO: methanol/HzO, methanol/DzO, and d4- shape of the deuterium spectra further interpretation and methanol/DzO. The proton spectrumfor "OH- plus meth- assignment of coupling constants is not possible at present. anol/HzO is shown in Fig. 30 (solid line); it has approximately ENDOR Spectra of "OH- in the Presence of Product- the same number of resonances as uncomplexed "OH- but The 'H and 'H ENDOR spectra at gl were examined for the the resonances are shifted to new positions. For example, the following three samples of "OH- plus 50 mM methanol in largest observed coupling is now AH = 7 MHz (resonance marked with an asterisk), decreased from 7.8 MHz in uncom- plexed "OH-. The superhyperfine constants atgl are given in Table I. For "OH- plus methanol/DzO (Fig. 30, dashed line), the intensities of at least three pairs of proton reso- nances are reduced. The difference spectrum shown in Fig. 3C is similar to that of uncomplexed "OH-, Fig. 3B. The resonances and AH values of these protons are noted with > arrows in Fig. 3C and with "ex" in Table I.5 The exchange of F protons for deuterons near the iron site also gives rise to the 'X -0 'H signal shown in Fig. 4B; it is similar in shapeand intensity to thespectrum of uncomplexed "OH- in Fig. 4A. If any of the remaining proton resonances in the ENDOR spectrum of"OH- plus methanol/D20 (Fig. 30, dashed line) originate from the methyl protons of methanol, then these should disappear for "OH- plus d4-methanol/Dz0, and new deuterium resonances should be observed. However, no suchchanges are observed in either theproton (not shown) IIIIIIIII or deuterium (Fig. 4, B and C)spectra. -4 -2 2 4 0 ENDOR Spectra of "OH- in the Presence of MezSO- VH (MHz) v - The proton ENDOR spectra at gl = 1.94 and g3 = 1.80 of FIG. 3. Proton ENDOR spectra at g, of "OH- in 50 mM "OH- plus 0.3 M MezSO are shown in Fig. 5, and the methanol and in D20. A, comparison of the ENDOR spectra of superhyperfine constants are given in Table I. Some of the "OH- in 100 mM MOPS, pH/pD 7.0, HzO (solid line) and DzO proton resonances also obey the empirical relation A$ = (dashed line) after 8 h of incubation. D, as in A except with 50 mM methanol. The difference spectra HzO-DzO are shown in traces B 0.9 AS as noted in Table I. Fig. 5 shows a pair of resonances (withoutmethanol) and C (withmethanol). The lower frequency at v - vH < -6 MHz, which have no partners at u - vH > +6 resonances of the exchangeable protons are indicated with an arrow MHz; they can be assigned to nitrogen and will be discussed in B and C. The spectra are all normalized to thesame vertical scale. Instrumental parameters: temperature, 10 K; microwave power, 20 The loss of signal amplitude of the associated resonance in DzO milliwatts at 9.37 GHz; gain, 1 X lo6; time constant, 0.1 s; 50 scans; of Fig. 30 (due to a broadened resonance) was not observed in a rf sweep rate, 0.2 MHz/s; frequency modulation, f25 kHz at 12.5 second sample. Thus, the proton with AH = 7 MHz does not appear kHz; rf attenuation, 6 dB. vH = 14.7 MHz. to be exchangeable. ENDOR of Methane Monooxygenase Hydroxylase Component 265
IIIIIIIII
IIIIIIIII ) -6 -2 2 6 I 13579 I1 v - VH (MHz) v(MHz) FIG.5. Proton ENDOR spectra for "OH- in 0.3 M MezSO. FIG. 6. 14N ENDOR spectra of "OH- and semimethem- A, gl= 1.94, pH = 14.88 MHz; B,g3= 1.80, vH = 16.02 MHz. Resonance erythrin sulfide. A, "OH- in 100 mM MOPS, pH 6.5, with 0.3 pairs for protons with large AHvalues are indicated with arrows. The M Me2S0,g, = 1.94, uN = 1.06 MHz; E, "OH- in 100 mM MOPS, pair of nitrogen resonances marked "N at g, is not observed at g3. pH 7.0, gl = 1.94; C, semimethemerythrin sulfide, pH 8, g, = 1.88, vN The off-resonance base line is flat and the spectra arenormalized to = 1.11 MHz. The spectra are normalized to the same vertical scale the same vertical scale. The sample was prepared in 100 mM MOPS for a concentration of 1 mM,except the semimethemerythrin sulfide buffer, pH 6.5. Instrumental parameters: temperature, 10 K, micro- spectrum hasbeen magnified by a factor of 2. The burs labeled Fe(iii)- wave power, 40 milliwatts at 9.463 GHz; gain,6.3 X lo6;time constant, N mark the resonance assignments for nitrogen coordinated to the 0.1 s; 10 scans; rf sweep rate, 0.4 MHz/s; frequency modulation, +50 ferric site of the diiron pair. The off-resonance base line has been kHz at12.5 kHz; rf attenuation, 10 dB. subtracted. Instrumental parameters: temperature, 10 K; microwave power, 20-50 milliwatts at 9.4 GHz; gain, 1 X lo6; time constant,0.1- 0.2 s; 15-30 scans; rf sweep rate, 0.2-0.5 MHz/s; frequency modula- below. The proton ENDOR spectrashow three pairs of reso- tion, +50 - +lo0 kHz at12.5 kHz; rf attenuation, 6-10 dB. nances from protonswith large AH values that were not observed for uncomplexed "OH-; these are marked with resonancesvanish at g3, leavinga broad signal valley for arrows in Fig. 5. Protons with large AHvalues are expected to frequencies v < 4 MHz (not shown),similar to that observed be very close to an iron. The resonance pair with AH = 7.8 at g, in Fig. 6A. This broad valley is only observed for fields MHz (noted with an asterisk) is also observed for uncom- within the EPR spectrum; for fields off EPR resonance the plexed "OH- (see Fig. 2A). At g3, the low frequency reso- ENDOR spectrum is flat. For a field 10 millitesla lower than nances for twoof the noted protons arein the samefrequency that of Fig. 6A, the broad valley is present but the sharp region and splitby nearly the samefrequency as the nitrogen features for v > 5 MHz disappear, indicating that the sharp resonances of Fig. 5A. However, this is coincidental since the features are not associated with the broad valley. The broad high frequency partners for the proton resonances areclearly valley, also observed in ENDOR spectra of uteroferrin, may visible. Thethree new pairs of resonances from strongly originate from remote nitrogens(17, 24). No other featuresat coupled protons are also observed for "OH- plus perdeu- either low frequency or in the higher frequency proton region terated MezSO (d6-MezSO), hence they do not originatefrom can be ascribed to I4N for "OH- plus Me2S0. the MezSO methyl protons (data notshown). Comparison of The nitrogen resonances of uncomplexed "OH- are the proton signals for "OH- with MezSO or d6-MezS0 did shown in Fig. 6B. One setof resonances with broader features not show any obvious differences that could be assigned to is at the same position (AN = 13.6 MHz, PN= 0.7 MHz) as the methylprotons. However, "OH- plus d6-Me2S0 in that of "OH- plus Me2S0.In addition, a new set of HzO gives rise to the deuterium signalshown in Fig. 40, resonances is observed in the lower frequency range of 2-5 indicating that Me2S0 closeis to the diiron cluster. MHz. The location and grouping of this lower set of reso- 14NENDOR Spectroscopy-The ENDOR spectrum from nances is not obvious; however theseresonances occur at I4N (I = 1) consists of four transitions at frequencies v = frequencies typical of nitrogen and they are distinctfrom the IAN/2 It vN k 3PN/21, where ANis the superhyperfinecoupling broad valley of Fig. 6A. At g3,both thelow and high frequency constant, vN is the nitrogen Larmor frequency (1.08 MHz for sets of resonances disappear leaving only a broad valley (not 350 millitesla) and PNis the quadrupole constant(23). Since shown) similar to thatof Fig. 6A. uN is known, the spectra are mosteasily interpreted by iden- For "OH- plusmethanol, the highfrequency set of tifying pairs of nitrogen resonances splitby 2vN; the coupling resonances is observed in the same position(AN = 13.6 MHz, constants ANand PNare then determinedfrom these pairings. PN = 0.7 MHz, not shown) and are as broad as thoseof Fig. The low frequency ENDOR spectrumof "OH- plus Me2S0 6B. Thesignal-to-noise of thespectra was inadequateto at gl has three distinct resonances shownas in Fig. 6A. These determine whether the low frequency set of resonances are resonances are notassociated with protons since, as shown inpresent. Fig. 5A, no high frequency proton partners areobserved. The 14NENDOR of Semimethemerythrin Sulfide-For compar- expected four line pattern isreduced to three lines due to an ison with thoseof "OH-, ENDOR spectraof semimethem- overlap of two resonances at v = 6.8 MHz. Each consecutive erythrin sulfide were recorded undersimilar instrumental pair of resonances is split by 2vN as indicated in Fig. 6A; the conditions. The EPR spectrum of the sample was homoge- I4N constants determined from this group of resonances are neouswith g values at gl = 1.87, g, = 1.69, g, = 1.41 as AN = 13.6 MHzand PN = 0.7 MHz. Thesharp nitrogen previously described (19,20). The nitrogen ENDOR spectrum 266 ENDOR of Methane MonooxygenaseHydroxylase Component at gl is shown in Fig. 6C. The overall pattern of resonances is from an interaction with theFe(I1) site, in which case Equa- similar to thatobserved for uncomplexed "OH-. The high- tion l'' gives A2 = 10.2 MHz. In either case, these intrinsic est frequency resonances can possibly be grouped as shown A? values are comparable to those previously measured from in Fig. 6C, giving coupling values of AN = 12.1 MHz and PN iron ligated nitrogens inmyoglobin (25) and theRieske center = 0.7 MHz. The lowest frequency resonance (u = 3.5 MHz) (26,27) (see Table11). They exceed the largestpossible dipolar overlaps with other resonances. contribution, which is 5 1.5 MHz according toEqua- tion 2 for a closest approach Fe-N distanceof r = 2 A. Hence, DISCUSSION theintrinsic Af;' value isdominated by the contact term, The mixed valence form of MMOHcontains high spin implying direct iron-nitrogenligation, thus providing the first direct identification of specific ligands to the diironcluster. ferric (S3= 5/2) and high spin ferrous (Sz= 2) iron atoms which are antiferromagneticallycoupled to give a ground spin Uncomplexed "OH- displays a second set of 14N reso- nances in the lower frequency range of2-5 MHz. Coupling state of S = 1/2. This spin doublet is the origin of the EPR constants for this lower frequency set of resonances cannot signals of Fig. 1. Relative to magnetically isolated irons, the presently be determined unambiguously, but it is reasonable antiferromagnetic exchange interaction reduces the spin ex- to assume that thevalue AH/2 is not greater than the centroid pectation value of the individual iron sites, where = 2,
Protein AN AqN AqN PN Refs. MHz "OH- 5.8 13.6 0.7 This work 57a 55.3 Semimet-HemerythrinS 12.1 5.2 This work 0.7 10.6 4.5 0.7 17 0.7 Uteroferrin*4.5 10.6 Met-myoglobin (0.3, 11.6)' (8.3, 8.1, 0.8, -1.1)' 25 Rieske Center of Phthalate Dioxygenased (0.9,4.3)2.9,(2.5, 0.5, 26-1.3) (3.4,3.7, 5.2) (0.8, 0.4,-1.2)
a This nitrogen may not be from histidine. * Mixed valence state of the diiron cluster. Valuesgiven here are determined by analysis of the published spectra (17). e The values in parenthesesare the coupling constants along the principal axis directions. For the w-(H/R)-oxo diiron proteins, only the magnitude of one principal axis value has been measured. Reduced state of the diiron cluster. TheA? values are calculated for the two 14N from Table I of Ref. 26. frequency set of nitrogen resonances at AN = 13.6 MHz in data canprovide a measure of the variations in theexchange both complexed and uncomplexed samples suggests that the interaction. If the value of J changes, then the ANvalue will coordination andgeometry of the Fe(II1) site is largely unal- also change, barring compensationby changes in eitherA: or tered by the MezSO interaction. In contrast, thelow frequency D;. The high frequency set of nitrogen resonances isobserved nitrogen resonances are no longer observed: suggesting that with the samecoupling constant AN= 13.6 MHz for "OH- the Fe(I1) site has undergone substantial alteration. Theloss in water, methanol, andMezSO. This lack of change suggests of the ENDOR signal is consistent with the displacementof that these inhibitors do not appreciably affect the exchange a nitrogen ligand(s) from the Fe(I1) site upon addition of interaction, therefore the electronic configuration of the p- Me2S0, but it is alsoconceivable that a significant change in (H/R)-oxo-bridge is also not appreciably changed. geometry at this sitecould result in the loss of the ENDOR 'H and 'H ENDOR Spectroscopy-Assessment of the pro- signal withoutligand displacement. ton ENDOR spectra can potentially yield information con- Recently, we have shown that the component B of the cerning at least four aspects of the active site structure:1) the MMO system forms a complex with the MMOH a-subunit accessibility of water to the active site, 2) the identification (10) essentialfor rapid turnovercoupled to NADH. The EPR of ligands with protons coupled to the diiron cluster, 3) the spectra of the "OH- and "OH2- states in thiscomplex distance of the coupled protons from a given iron, and 4) the are significantly different than thoseof uncomplexed MMOH. extent of interaction of small molecules with the active site. In contrast, the reductase component binds to the MMOHp- Someinsight into each of theseaspects accruesfrom the subunit without alterationof the lineshape of the EPRspec- current study asdiscussed below. tra. This suggests that the diiron cluster islocated in the a- The active site of "OH- is accessible to water asshown subunit. Accordingly, theamino acidsequence of the a- by the fact thatat least threeof the ninemagnetically distinct subunitdetermined from the nucleotidesequence of the coupled protons can be exchanged for deuterium. However, cloned gene (30) contains twosequences with stronghomology over an 8-h period these protons have exchanged in only a to theiron bindingregions of the diiron clusterof Escherichia portion of the molecules in the sample. The incomplete ex- coli ribonucleotide reductase B2 subunit (31, 32). Each of change suggests that water access to the site is restricted. In these sequences contains a single histidine,His147a and contrast, completeexchange of the exchangeable protons near His246a. This is consistent with our observationof nitrogen the diiron sitesof ribonucleotide reductase, hemerythrin (36), ligation at each iron of "OH- and suggests that the nitro- and uteroferrin isobserved for incubation times significantly gen at each ironmay derive from histidine. The complete loss less than 8 h.7 of nitrogen ENDOR associated here with the Fe(I1) site on The nitrogen ENDOR results presented here demonstrate addition of MezSO is also consistent with the presence of a the presence of at least one and probably two histidine iron single nitrogen ligand at theFe(I1) site, but asdescribed above ligands. Thus,it is reasonable that at least some of the other explanationsfor the loss of the signal are possible. exchangeable protonsare associated withthe nonbonding Thevariation of Fe(II1)-Ncouplings A: inTable I1 nitrogens of these ligands. Conversely, the A values of the (MMOH > hemerythrin > uteroferrin) is presumably due to exchangeable protons support the presence of histidines in breakdown of the assumption J >> D; used in Equation 1, the cluster coordination. An estimate of the maximum value where D; are the zero-field splittings for each iron. Including of the superhyperfine coupling for a proton can be made by a first order correction gives AN = (7/3)Ay(1 + (4/15)(8D3 + assuming that the histidine coordinates only to one iron' and 3Dz)/J) (9). Accurate values for Di are not yet available for that the magnitude of the superhyperfine constant is due these proteins, but we expect roughly Dz = 5 cm" and D3 z 0.5 cm" (33-35). For uteroferrin (J = -9.9 cm" (35)), the Although the exchange kinetics have not been reported, typical correction to thevalue of A: can be 30%, which is of the order incubation periods necessary for proton exchange in these proteins of magnitude of the variations in A:. The correlation of the are less than 1 h. T. Elgren and L. Que, Jr., personal communication. nitrogen superhyperfine constant AN with the exchangecou- 'For certain orientations of the molecule with respect to the pling J is importantfor the present studybecause the nitrogen magnetic field direction, the dipolar contribution to AH from the coupling of a proton to the more distant iron of the pair can be large, thus invalidating the assumption. Currently, the lack of information The proton frequency region was checked carefully for resonances regarding the orientation of the g tensor of "OH- with respect to which had no proton partner; no evidence for such a resonance was the molecule makes determination of the relative dipolar contribu- observed. tions impossible. 268 ENDOR of Methane Monooxygenme Hydroxylase Component solely to the magnetic dipole intera~tion.~ Theexchangeable the EPRspectrum of "OH-. For the Me2S0complex, new proton fqr histidine coordination is-typically at a distance of proton ENDOR resonances are observed with significantly 4.9-5.3 A from Fe(II1) or 5.2-5.8 A from Fe(I1) (29). From larger superhyperfine couplings than those observed for the Equations 1 and 2, a proton 5 A from the Fe(II1) or Fe(I1) uncomplexed "OH-. These new resonances (A: = 6.5 site gives AH 5 2.9 MHz or AH 5 1.7 MHz, respectively. Thus, MHz, A; = 9.1, 13.0 MHz) do not derive from the methyl the AH values of the exchangeable protons are compatible protons of Me2S0 since they are also observed in d6-Me2S0, with assignment to theexchangeable proton of a histidine. nevertheless, d6-MepSO does bind close to thediiron site since A consistent feature of the proton spectra in water, meth- deuterium resonances are observed. The close proximity of anol, and Me2S0is the conserved pair of resonances with AH Me2S0 is presumably responsible in part for the dramatic = 7-7.9 MHz at gl. The magnitude of the coupling indicates EPR spectral changes. This is thefirst evidence for any that theproton associated with this resonance pair is close to molecule binding near the active site cluster of "OH. an iron and probably associated with an iron ligand. The Correspondence between the ENDOR Resonances at gl and proton is not exchangeable with deuterium and also is not g3-A correlation between several of the proton ENDOR appreciably affected by the presence of Me2S0suggesting that resonances at gl and g3 was noted under "Results," namely, it is associated with the Fe(II1) rather than the Fe(I1) site. A$ = 0.9AZ. The validity of this empirical correlation is Given the probable presence of a histidine ligand(s) on the strengthened by the observation that two of the correlated Fe(II1) site, candidates for this proton(s) are those on the resonances exhibit equivalent decreases in intensity upon carbons of the imidazole ring closest to Fe-N.A typical deuteration. In the absence of specific isotopic substitutions distance for a protonof this type from Fe(II1) is approximately of protons near the active site, the empirical correlation is 3.4 A (29) giving AH 5 9.4 MHz from Equations 1' and 2, in useful in theidentification of specific proton resonances which agreement with the value observed experimentally. shift due to changes in protein ligation or environment. We The relatively weak exchange coupling (3) and the apparentcurrently have no quantitative explanationfor this correlation lack of a short Fe-p-oxo bond (2) observed for MMOH are which hasnot been reported for other proteins. We have consistent with a protonated or otherwise substituted oxo- checked for such a correlation in the proton resonances of bridge (38). Aproton in this position would probably be semimethemerythrin sulfide; it was not observed. The factor exchangeable. The close distance would very likely give a 0.9 can possibly be attributed to the anisotropy of the elec- large contact contribution to the superhyperfine coupling in tronic environment of the diiron cluster, that is
The assumption provides areasonable startingpoint for the determination of proton distances (37),but itis approximate because it ignores the electronic contact contribution. For example, the NH proton of Fe(II1)-His in uteroferrin has a paramagnetic shift of 90 ppm (300 MHz, 31 "C) (29). If this shift is solely due to a contact contribution, then under the conditions of a typical ENDOR experi- ment Aeontact= 0.5 MHz (3500 G, 10 K), which is 40% of the largest possible dipolar contribution. SCHEMEI. ENDOR of Methane Monooxygenme Hydroxylase Component 269 bearing oxo-bridged diiron clusters and the similarity of the 18. Fox, B. G., Froland, W. A., Jollie, D. R., and Lipscomb, J. D. nitrogen ENDOR spectrum observed with that of semime- (1990) Methods Enzymol. 188, 191-202 themerythrin sulfide. The apparent displacement or reorien- 19. Kurtz, D. M., Jr., Sage, J. T., Hendrich, M. P., Debrunner, P. G., and Lukat, G. S. (1983) J. Biol. Chem. 258, 2115-2117 tation of this ligandupon binding MeZS0 near the is 20. Lukat, G. S., andKudz, D.M., Jr. (1985) Biochemistry 24,3464- unprecedented in spectroscopic studies of p-oxo-bridged din- 3472 uclear iron clusters and may be related to the unique aspects 21. Fox, B. G., Froland, W. A., and Lipscomb, J. D. (1990) in Gas, of the cluster in MMOH which allows catalysis of oxygenase Oil and Coal Technology I (Akin, C. and Smith, J.,eds) pp. 197- chemistry. 214, Institute of Gas Technology Press, Chicago 22. Hoffman, B. M., Martinsen, J., and Venters, R. A. (1984) J. REFERENCES Magn. Reson. 59,110-123 23. Abragam, A., and Bleaney, 8. (1970) Eleetron Paramagnetic 1. Fox., B.G., Froland, W. A., Dege, J. E., and Lipscomb, J. D. Resonance of Transition Ions, Oxford University Press, New (1989) J. Biol. Chem. 264, 10023-10033 York 2. Ericson, A., Hedman, B., Hodgson, K. O., Green, J., Dalton, H., 24. Antanaitis, B. C., Peisach, J., Mims, W. B., and Aisen, P. (1985) Bentsen, J. G., Beer, R. H., and Lippard, S. J. (1988) J. Am. J. Biol. Chem. 260,4512-4574 Chem. SOC.110, 2330-2332 25. Scholes, C. P., Lapidot, A., Mascarenhas, R., Inubushi, T., Isaac- 3. Fox, B. G., Surerus, K. K., Munck, E., and Lipscomb, J. D (1988) son, R. A., and Feher, G. (1982) J. Am. Chem. SOC.104, 2724- J. Biol. Chem. 263,10553-10556 2735 4. Anthony, C. (1982) The Biochemistry of the Methyltrophs, Aca- 26. Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., demic Press, London Hoffman, B. M., and Ballou, D. P. (1989) Biochemistry 28, 5. Dalton, H. (1980) Adu. Appl. Microbiol. 26, 71-87 4861-4871 6. Higgins, I. J., Best, D. J., and Hammond, R. C. (1980) Nature 27. Britt, R. D., Sauer, K., Klein, M. P., Knaff, D. B., Kriauciunas, 286,561-564 A., Yu, C.-A., Yu, L., and Malkin, R. (1991) Biochemistry 30, 7. Hendrich, M. P., Munck, E., Fox, B. G., and Lipscomb, J. D. 1892-1901 (1990) J. Am. Chem. SOC.112, 5861-5865 28. Stenkamp, R. E., Sieker, L. C., and Jensen, L. H. (1984) J. Am. 8. Guigliarelli, B., Bertrand, P., and Gayda, J.-P. (1986) J. Chem. Chem. SOC.106, 618-622 Phys. 85, 1869-1692 29. Scarrow, R. C., Pyrz, J. W., and Que, L., Jr. (1990) J. Am. Chem. 9. Sage, J. T., Xia, Y.-M., Debrunner, P. G., Keough, D. T., de SOC.112,657-665 Jersey, J., and Zerner, B. (1989) J. Am. Chem. SOC.11 1,7239- 30. Cardy, D. L. N., Laidler, V., Salmond, G. P. C., and Murrel, J. C. 7247 (1991) Mol. Microbiol. 5, 335-342 10. Fox, B. G., Liu, Y., Dege, J. E., and Lipscomb, J. D. (1991) J. 31. Carlson, J., Fuchs, J. A., and Messing, J. (1984) Proc. Natl. Acad. Biol. Chem. 266,540-550 Sei. U. S. A. 81,4294-4297 11. Andersson, K. K., Froland, W. A., Lee, S.-K., and Lipscomb, J. 32. Nordlund, P., Sjoberg, B. M., and Eklund, H. (1990) Nature 345, D. (1991) New J. Chem. 15, 411-415 593-598 12. Que, L., Jr.,and True, A. E. (1990) in Progress in Inorganic 33. McCormick, J. M., and Solomon, E. I. (1990) J. Am. Chem. SOC. Chemistry, (Lippard, S., ed) Vol. 38, pp. 201-258, John Wiley 112,2005-2007 & Sons, New York 34. Beck, J. L., de Jersey,J., Zerner, B., Hendrich, M. P., and 13. Sanders-Loehr, J. (1989) in Iron Carriersand Iron Proteins Debrunner, P. G. (1988) J. Am. Chem. SOC.110,3317-3318 (Loehr, T. M., ed) pp. 373-466, VCH Publishers, New York 35. Day, E. P., David, S. S., Peterson, J., Dunham, W. R., Bonvoisin, 14. Vincent, J. B., Olivier-Lilley, G., and Averill, B. A. (1990) Chem. J. J., Sands, R. H., and Que, L., Jr. (1988) J. Biol. Chem. 263, Rev. 90,1447-1467 15561-15567 15. Woodland, M. P., and Dalton, H. (1984) J. Biol. Chem. 259,53- 36. Shiemke, A. K., Loehr, T. M., and Sanders-Loehr, J. (1984) J. 59 Am. Chem. Soc. 106,4951-4956 16. Dalton, H. (1980) Microbial Growth on C, Compounds, (Dalton, 37. Mulks, C. F., Scholes, C. P., Dickinson, L.C. and Lapidot, A. H., ed), pp. 1-10, Heyden Press, London (1979) J. Am. Chem. SOC.101, 1645-1654 17. Doi, K., McCracken, J., Peisach, J., and Aisen, P. (1988) J. Biol. 38. Armstrong, W. H.,and Lippard, S. J. (1984) J. Am. Chem. SOC. Chem. 263,5757-5763 106,4632-4633