Implications for Electron Transfer A

Faculty Scholarship

2004 Inhibition of Membrane-Bound Methane Monooxygenase and Ammonia Monooxygenase by Diphenyliodonium: Implications for Electron Transfer A. K. Shiemke

D. J. Arp

L. A. Sayavedra-Soto

Follow this and additional works at: https://researchrepository.wvu.edu/faculty_publications

Digital Commons Citation Shiemke, A. K.; Arp, D. J.; and Sayavedra-Soto, L. A., "Inhibition of Membrane-Bound Methane Monooxygenase and Ammonia Monooxygenase by Diphenyliodonium: Implications for Electron Transfer" (2004). Faculty Scholarship. 196. https://researchrepository.wvu.edu/faculty_publications/196

This Article is brought to you for free and open access by The Research Repository @ WVU. It has been accepted for inclusion in Faculty Scholarship by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. JOURNAL OF BACTERIOLOGY, Feb. 2004, p. 928–937 Vol. 186, No. 4 0021-9193/04/$08.00ϩ0 DOI: 10.1128/JB.186.4.928–937.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Inhibition of Membrane-Bound Methane Monooxygenase and Ammonia Monooxygenase by Diphenyliodonium: Implications for Electron Transfer Andrew K. Shiemke,1* Daniel J. Arp,2 and Luis A. Sayavedra-Soto2 Department of Biochemistry and Molecular Pharmacology, Robert C. Byrd Health Sciences Center, West Virginia University School of Medicine, Morgantown, West Virginia 26506-9142,1 and Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331-29022

Received 16 September 2003/Accepted 7 November 2003 Downloaded from

Diphenyliodonium (DPI) is known to irreversibly inactivate ﬂavoproteins. We have found that DPI inhibits both membrane-bound methane monooxygenase (pMMO) from Methylococcus capsulatus and ammonia monooxygenase (AMO) of Nitrosomonas europaea. The effect of DPI on NADH-dependent pMMO activity in vitro is ascribed to inactivation of NDH-2, a ﬂavoprotein which we proposed catalyzes reduction of the quinone pool by NADH. DPI is a potent inhibitor of type 2 NADH:quinone oxidoreductase (NDH-2), with 50% inhibition occurring at Ϸ5 ␮M. Inhibition of NDH-2 is irreversible and requires NADH. Inhibition of NADH-dependent pMMO activity by DPI in vitro is concomitant with inhibition of NDH-2, consistent with our proposal that NDH-2 mediates reduction of pMMO. Unexpectedly, DPI also inhibits pMMO activity driven by exogenous hydroquinols, but with Ϸ100 ␮M DPI required to achieve 50% inhibition. Similar concentrations of DPI are http://jb.asm.org/ required to inhibit formate-, formaldehyde-, and hydroquinol-driven pMMO activities in whole cells. The pMMO activity in DPI-treated cells greatly exceeds the activity of NDH-2 or pMMO in membranes isolated from those cells, suggesting that electron transfer from formate to pMMO in vivo can occur independent of NADH and NDH-2. AMO activity, which is known to be independent of NADH, is affected by DPI in a manner analogous to pMMO in vivo: Ϸ100 ␮M is required for 50% inhibition regardless of the nature of the reducing agent. DPI does not affect hydroxylamine oxidoreductase activity and does not require AMO turnover to exert its inhibitory effect. Implications of these data for the electron transfer pathway from the quinone pool to pMMO and AMO are discussed. on September 6, 2018 by guest

Ammonia monooxygenase (AMO) and the membrane- isolating pure samples of either enzyme with high activity (4, bound form of methane monooxygenase (pMMO) are two of 17, 28, 31, 49, 51). the three members of a novel family of membrane-bound AMO and pMMO initiate oxidative metabolism of NH3 and monooxygenases capable of oxidizing small hydrocarbons (3, CH4 in nitrifying and methanotrophic bacteria, respectively 33). These two enzymes are similar in their putative subunit (3, 19). Both types of bacteria obtain energy solely from the composition, inhibitor profiles, and the DNA sequence of the subsequent oxidation of the products of the monooxygenase genes encoding the subunits (19, 20, 22). The membrane- reaction. In methanotrophs, energy needs are satisfied by the bound butane monooxygenase from Nocardioides sp. strain oxidation of methanol to CO2 by periplasmic and cytosolic CF8 was recently identified as the third member of this class of dehydrogenases specific for methanol, formaldehyde, and for- enzymes, sharing many of the characteristics of pMMO and mate (1). In nitrifying bacteria, the 4-electron oxidation of AMO (18). These enzymes are known to require copper, be- hydroxylamine to nitrite by hydroxylamine oxidase (HAO) pro- cause inhibition by metal chelators is reversed only by addition vides reductant for ammonia oxidation and all other cellular of copper (14, 17). In addition, all three are irreversibly inhib- processes (8). ited by acetylene, and when radiolabeled acetylene is used, a AMO and pMMO are notoriously refractory to purification. Ϸ 27-kDa peptide is specifically labeled (5, 18, 26, 37). AMO has not been purified to homogeneity with activity, and Enzymes of this class can oxidize small alkanes and haloge- although several laboratories have reported purification of nated solvents, hence their potential application in bioreme- active pMMO (4, 31, 34, 49, 51), the specific activity of the diation (9, 25, 29, 45). Although there are other enzymes that purified enzyme is a small fraction of the activity in vivo. are able to oxidize small alkanes (32, 46, 47), they are distinct Disruption of the physiological electron transfer pathways to in structure and properties from AMO and pMMO. These two AMO and pMMO may be in part responsible for the deterio- enzymes thus represent a novel mechanism for alkane oxida- ration of activity in vitro (40, 41). These pathways are poorly tion, one that has yet to be elucidated due to the difficulty in characterized (1, 48), and thus, the reducing agents used in vitro may not be physiologically relevant, resulting in inefficient electron transfer to their active sites. * Corresponding author. Mailing address: Department of Biochem- NADH generated from oxidation of formaldehyde and for- istry and Molecular Pharmacology, Robert C. Byrd Health Sciences mate is believed to be the source of reducing equivalents for Center, P.O. Box 9142, West Virginia University School of Medicine, Morgantown, WV 26506-9142. Phone: (304) 293-2310. Fax: (304) 293- methane oxidation (2). It is well established that NADH di- 6846. E-mail: [email protected]. rectly reduces the soluble form of methane monooxygenase

928 VOL. 186, 2004 INHIBITION OF pMMO AND AMO BY DIPHENYLIODONIUM 929 Downloaded from

FIG. 1. Proposed electron transfer pathways to pMMO in M. capsulatus (A) and to AMO in N. europaea (B). The solid lines in A depict the pathway proposed in reference 13, and the dashed lines represent the NADH-independent pathway we propose for in vivo reduction of pMMO based on the data presented in this work. The pathway depicted in part B is adapted from reference 48. In both pathways, proposed sites of inhibition by DPI are indicated by a vertical bar.

(sMMO), mediated by an iron-sulfur flavoprotein reductase nism involving reduction of DPI and radical recombination http://jb.asm.org/ (32). NADH is also the optimal reductant for pMMO in iso- with the cofactor (16). lated membrane fractions (42). However, we and others have In this work we show that DPI irreversibly inactivated the reported that NADH is ineffective with detergent-solubilized NDH-2 from M. capsulatus, leading to irreversible inhibition of or purified pMMO, which can only be reduced by exogenous NADH-dependent pMMO activity in vitro, as expected (solid hydroquinols, such as duroquinol (4, 40, 41, 51). We recently lines in Fig. 1A). However, DPI inhibited pMMO activity in reported isolation of a 36-kDa flavoprotein from membranes vitro even when reducing equivalents were provided down- of Methylococcus capsulatus that has characteristics of a type 2 stream of NDH-2 by hydroquinols, suggesting that a second NADH:quinone oxidoreductase (NDH-2) and suggested that DPI-sensitive protein is required for reduction of pMMO. We it mediates electron transfer to pMMO in conjunction with the show that DPI also inhibited pMMO activity in whole cells in on September 6, 2018 by guest endogenous quinone pool (solid lines in Fig. 1A) (13). Support a manner that is apparently independent of NDH-2 (dashed for this suggestion comes from a recent report in which high lines in Fig. 1A). DPI irreversibly inhibited the activity of pMMO activity was sustained by NADH and exogenous qui- AMO in whole cells of Nitrosomonas europaea as well. nones only after addition of purified NDH-2 (11). Further- Electron transfer to AMO is independent of NADH (Fig. more, the specific activity in the presence of duroquinone, 1B) (22, 48), so the site of DPI inhibition cannot be analogous NADH, and NDH-2 was Ϸ40% greater than that with duro- to the NDH-2 of M. capsulatus. AMO is inhibited by DPI quinol alone (11). However, Chan and coworkers recently re- whether reducing equivalents are donated through HAO by ported pMMO activity in detergent-solubilized membranes hydroxylamine or hydrazine or at the level of the quinone pool with either NADH or duroquinol as the reductant but 10-fold by duroquinol. Furthermore, DPI does not affect electron greater specific activity with the quinol (49). Their purified transfer from HAO to the terminal oxidase and does not have pMMO samples had similar specific activity with either NADH the characteristics of a mechanism-based inhibitor of AMO. or duroquinol as the reductant (49), but 4- to 10-fold lower Thus, we propose that DPI acts to interrupt electron transfer than reported by others (4, 11). Clearly, more work is necessary from the quinone pool to AMO (Fig. 1B), perhaps by irrevers- to elucidate electron transfer to pMMO both in vivo and in vitro. In the present study we used diphenyliodonium (DPI) to further investigate the electron transfer pathways to pMMO and AMO. DPI and other diaryl-iodonium compounds irreversibly inactivate many redox-active proteins, primarily flavoproteins (10, 15, 35, 36, 43), but DPI may also react with some low-potential b-type cytochromes and quinoproteins (6, 16). The proposed mechanism for DPI inactivation of flavoproteins (10, 35) is shown in Fig. 2: DPI accepts one electron from the fully reduced flavin, generating a DPI free radical and the flavin semiquinone. Spontaneous cleavage of the DPI radical yields a benzene radical which combines with the flavin semiquinone, resulting in covalent modification of the cofactor FIG. 2. Proposed mechanism for inhibition of flavoproteins by and irreversible inactivation of its redox cycle. DPI is thought DPI, adapted from references 10 and 34. Fl represents the protein- to inactivate other redox-active proteins by a similar mecha- bound flavin cofactor. 930 SHIEMKE ET AL. J. BACTERIOL.

ibly binding to a site analogous to the NDH-2-independent site change in percent oxygen saturation over time was recorded on a chart recorder. The rate of oxygen uptake in micromoles per minute was calculated based on a in M. capsulatus. ␮ concentration of 230 MO2 in air-saturated buffer (44). HAO activity was also measured polarographically as the rate of hydroxy- MATERIALS AND METHODS lamine-dependent oxygen uptake (30). Samples were prepared as above, and the ␮ Growth of bacteria. M. capsulatus (strain Bath) was maintained at 45°Con assay was initiated by adding 10 l of a 0.25 M solution of hydroxylamine. To 1.8% agar plates in an atmosphere of Ϸ30% methane in air, and cultures in determine AMO and HAO activity on the same sample, AMO activity was Ϸ liquid medium were grown at 45°C in NMS medium supplemented with phos- measured as above, but when the oxygen concentration reached 40% satura- ␮ phate buffer, vitamins, and copper, as described in previous reports (13, 40). M. tion, 10 l of 100 mM allylthiourea was added to completely inhibit O2 uptake capsulatus was harvested at late log phase by centrifugation for 10 min at 8,000 by AMO. HAO activity was then measured by adding hydroxylamine, as de- ϫ g from either 500-ml batch cultures in 2-liter flasks or 10-liter batch cultures scribed above. grown in a 14-liter fermenter according to published procedures (40). Cells were NDH activity of membrane fractions and purified NDH-2 was measured as washed one to three times in a volume of 10 mM piperazine-N,NЈ-bis(2-ethane- either the rate of quinone reduction in the presence of NADH or the rate of sulfonic acid) (PIPES, pH 7.2) equal to 2% of the culture volume. The washed quinone-dependent NADH oxidation, according to methods described previ- cells were suspended in 50 mM PIPES, pH 7.2, in a final volume that was 0.5% ously (13). The rate of absorbance change was measured with an Aminco DW- 2000 spectrometer, equipped with a stirred, thermostatted cell holder.

of the culture volume. Cells were stored at 4°C under argon and typically used Downloaded from within 5 days of harvesting. In some cases the effect of DPI on enzyme activity was determined by per- N. europaea was grown in batch cultures at 30°C with shaking in a minimal forming the assay in the presence of the inhibitor (as in Fig. 3 and 5A), which was medium, as previously described (17, 29). The cells were harvested by centrifu- prepared as a 10 mM stock solution in aqueous buffer. In these assays the gation at 8,000 ϫ g from 1.5-liter cultures at late log phase. Cells were washed presence of DPI and reductant caused the enzyme activity to decrease steadily three times with 50 mM phosphate, pH 8.0, and suspended in the same buffer at during the assay. Therefore, activity was determined from the initial linear a volume equivalent to 0.1% of the culture volume. Cells were stored at 4°C and portion of the assay: the ﬁrst 60 s for NDH-2 and the ﬁrst 3 min for pMMO. In used within 3 days. all other cases the cells or membranes were incubated with DPI, followed by Membrane isolation. Similar procedures were used to isolate membrane frac- extensive washing to remove the unreacted inhibitor prior to performing the tions from M. capsulatus and N. europaea. Cells were suspended in buffer (50 mM assays, as described above. PIPES, pH 7.2, for M. capsulatus and 50 mM phosphate, pH 8.0, for N. europaea) Protein concentrations were determined with the bicinchoninic acid assay with

at 15 to 30 mg/ml total protein concentration. Both types of cell suspension were reagents from Pierce Chemical Corp. and bovine serum albumin as the standard. http://jb.asm.org/ ␮ Whole cells and membrane fractions were digested in 1.0 M NaOH at 90°C for supplemented with 1 mM phenylmethylsulfonyl ﬂuoride and 200 M Cu(SO4), and N. europaea also contained 10 mg of bovine serum albumin per ml. Cell 10 min prior to the protein assay. suspensions were passed three times through a French press cell at 18,000 to Materials. Ethanol was removed from NADH by two cycles of dissolving it in 20,000 lb/in2. The cell lysate was centrifuged at 8,000 ϫ g for 10 min to remove water and lyophilizing to dryness. Quinones and hydroquinols were prepared as Ϸ unbroken cells. Sufﬁcient NaCl was added to the supernatant to bring the con- concentrated stock solutions ( 50 mM) in ethanol or dimethyl sulfoxide. The centration to 1 M, to facilitate release of peripheral proteins from the membrane. dimethyl sulfoxide solutions were added directly to the protein samples. The The supernatant was then centrifuged at 106,000 ϫ g for 60 to 90 min. The pellet quinones in ethanol solution were placed in a vial and dried under vacuum to was suspended with a Dounce homogenizer in 50 mM buffer containing 100 ␮M remove the solvent to prevent interference with GC detection of assay products. ϫ The protein sample was added to this vial and vortexed vigorously prior to Cu(SO4), centrifuged again at 106,000 g for 60 min, and suspended in a volume

of 50 mM buffer equivalent to half of the original volume of cell suspension. addition of the substrate. on September 6, 2018 by guest Membrane samples were stored at 4°C and used for experiments within 48 h of Hydroquinols were prepared by reducing the quinone with zinc dust in either isolation. ethanol or dimethyl sulfoxide containing 50 mM HCl. For a typical reaction, 1 ml Ϸ Detergent solubilization of pMMO and NDH-2 and purification of NDH-2. of quinone ( 50 mM in ethanol or dimethyl sulfoxide) was placed in a 1.5-ml ␮ NDH-2 and pMMO were solubilized from the M. capsulatus membrane fraction microcentrifuge tube, and 20 mg of Zn dust was added, followed by 50 lof1.0 by addition of a 20% (wt/vol) stock solution of lauryl maltoside. The detergent M HCl. This sample was shaken at room temperature until it became colorless. ϫ was added with vortexing to a final detergent-to-protein ratio of 0.9 (wt/wt). The The excess Zn dust was removed by centrifugation at 14,000 g for 3 min. 2ϩ sample was incubated on ice for 30 to 45 min with frequent vortexing, then Water-soluble contaminants (principally Zn ) were removed by adding the centrifuged at 106,000 ϫ g for 60 min. The supernatant was used immediately for supernatant to 15 ml of 10 mM HCl. The hydroquinol formed a white precipitate pMMO or NDH assays or NDH-2 isolation. The NDH-2 was purified by column that was collected by centrifugation, washed twice with 10 mM aqueous HCl, and Ϸ chromatography, and purity was determined by silver-stained sodium dodecyl dissolved in 0.5 ml of ethanol or dimethyl sulfoxide containing 10 mM HCl. Ϫ sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, as described pre- The stock solutions of hydroquinol were stored at 80°C to prevent oxidation. viously (13). The purified protein was stored at 4°C and used for experiments The concentrations of quinone and hydroquinol stock solutions were determined within 1 week. No loss of activity was observed over this time. spectrophotometrically, with published extinction coefficients (40). All other Enzyme assays. The activity of pMMO was assayed by measuring the rate of materials were used as supplied by the manufacturer without further purification. propylene oxidation, as previously described (12, 13). Propylene oxide was measured by injecting either 2 ␮l of solution or 100 ␮l of headspace into a SRI-8610 gas chromatograph (SRI Instruments, Torrance, Calif.) equipped with an Rtx-1 RESULTS capillary column (30 m by 0.53 mm; Restek Corp., Bellefonte, Pa.) and flame ionization detector. The concentration of propylene oxide was determined from DPI inhibition of NDH-2. NDH-2 from M. capsulatus cata- the integrated peak area relative to a standard curve. Formate, formaldehyde, or lyzes NADH-dependent quinone reduction (13). DPI was a duroquinol was used as the reductant for whole-cell assays, as indicated in the potent inhibitor of NADH-dependent quinone reduction in M. figure legends, whereas NADH or hydroquinols were used to assay membrane and detergent-solubilized samples, according to previously published methods capsulatus membranes (Fig. 3). This inhibition required reduc- (13, 40). tion by NADH; longer incubation of the membrane samples AMO activity was determined from the rate of ethylene oxidation or the rate with DPI and NADH (0, 3, and 8 min) resulted in a dramatic of ammonia-dependent oxygen uptake (27, 38). In the former assay, 1 ml of increase in the extent of inhibition (Fig. 3). In contrast, little or sample was incubated at 30°C for 3 min in a septum-sealed 6-ml vial. The assay no effect on quinone reductase activity was observed after was initiated by adding 5 to 10 ␮l of reductant (hydrazine, hydroxylamine, or duroquinol) from a concentrated stock solution, followed by 3 ml of ethylene. incubating membranes for 8 min with DPI in the absence of Ethylene oxide concentration was measured by injecting 100 ␮l of headspace into NADH (Fig. 3). Incubation of purified NDH-2 for 45 s with 10 a Shimadzu model GC-8A gas chromatograph (Shimadzu Corp., Kyoto, Japan) ␮M DPI and 200 ␮M NADH results in Ͼ97% inhibition of the equipped with a Porapak-Q column (0.3 cm inner diameter by 40 cm long; rate of duroquinone-dependent NADH oxidation (Fig. 3, in- Waters Associates, Framingham, Mass.) and flame-ionization detector and ref- erenced to a standard curve. The oxygen uptake assay was performed with a set). The effect of DPI on NDH-2 activity was irreversible; polarographic oxygen electrode (Yellow Springs Instruments) and a cell main- overnight dialysis of purified, DPI-treated NDH-2 resulted in ␮ tained at 30°C. The assay was initiated by adding 6 lof1M(NH4)2SO4 and the no change in the extent of inhibition (data not shown), and VOL. 186, 2004 INHIBITION OF pMMO AND AMO BY DIPHENYLIODONIUM 931 Downloaded from http://jb.asm.org/

FIG. 3. Inhibition of NDH-2 from M. capsulatus by DPI is enhanced by incubation with NADH. Membrane samples were incubated at 25°C with 0to25␮M DPI and 1 mM NADH for 0, 3, or 8 min. The assay was initiated by addition of 1 mM 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q- zero), and the rate of Q-zero reduction was monitored by the decrease in absorbance at 410 nm during the initial 45 s of the assay. As a control, membrane samples were incubated for 8 min with 0 to 25 ␮M DPI in the absence of NADH before initiating the assay. Each data point is reported as the mean value Ϯ standard deviation of three or four measurements. The inset shows the effect of 10 ␮M DPI on the activity of pure NDH-2. DPI and 0.2 mM NADH were added to the enzyme sample at time zero and incubated at 25°C for 45 s before initiating NDH-2-catalyzed NADH oxidation with 0.25 mM Q-zero.

extensive washing of DPI-treated membranes did not reverse creased availability of the reduced ﬂavin to DPI. Decyl- on September 6, 2018 by guest the inhibitory effect of DPI (Fig. 4). plastoquinone (PQ) is an effective electron acceptor for NDH- DPI inhibition of pMMO. To determine if DPI affects 2 (13), and therefore we examined the ability of PQ to protect pMMO activity, membrane samples from M. capsulatus were pMMO from DPI inhibition. Increasing concentrations of PQ incubated with increasing concentrations of DPI in the presence of 2 mM NADH. The results of this titration with DPI (Fig. 4) demonstrated that NADH-driven pMMO activity was extensively inhibited by DPI. Only Ϸ8% of the original pMMO activity remained after treatment with 50 ␮M DPI. Measure- ment of NDH-2 activity in the same DPI-treated membrane samples demonstrated a comparable response to DPI, such that 50% inhibition was observed at Ϸ2 ␮M and Ϸ5 ␮M for NDH-2 and pMMO activities, respectively (Fig. 4). In this experiment, the activities of both NDH-2 and pMMO were measured after the membranes were washed to remove unreacted DPI and NADH, suggesting that the effect of DPI is irreversible. When NADH was absent from the incubation with DPI, little or no inhibition of either NDH-2 or pMMO activity was observed (data not shown). As was observed for NDH-2 activity, the extent of DPI inhibition of pMMO activity depended on the length of the incubation with NADH (Table 1). In this experiment, the effect FIG. 4. DPI inhibition of pMMO and NDH-2 in membranes from of DPI was determined without washing the membranes. M. capsulatus. Membrane samples were incubated for 3 min at 45°C ␮ Therefore, signiﬁcant inhibition is observed for the time zero with 2 mM NADH and 0 to 50 M DPI and washed three times by centrifugation before measuring pMMO and NDH activity. The incubation, since DPI and NADH are present throughout the pMMO activity was measured as the rate of NADH-driven propylene assay (Table 1). epoxidation. NDH activity was measured as the rate of NADH oxida- Some DPI-sensitive enzymes are protected from inactivation tion in the presence of 1 mM duroquinone and 125 ␮M NADH. Ac- tivities are expressed as a percentage of that of the 0 ␮M DPI controls in the presence of an electron acceptor. For example, increas- Ϫ Ϫ Ϫ Ϫ (154 nmol min 1 mg 1 for pMMO and 930 nmol min 1 mg 1 for ing concentrations of cytochrome c resulted in reduced sensi- NDH-2). Each data point is reported as the mean value Ϯ standard tivity of cytochrome P450 reductase to DPI, ascribed to de- deviation of three measurements. 932 SHIEMKE ET AL. J. BACTERIOL.

TABLE 1. Effect of incubation time on DPI inhibition of pMMO (Fig. 5B), whereas the NADH-dependent in vitro activities a activity in membranes and protection by PQ required less than 5 ␮M DPI to reach 50% inhibition (Fig. 4). Mean pMMO sp act To determine if the effect of DPI on whole-cell pMMO Ϫ Ϫ Incubation conditions (nmol min 1 mg 1) Ϯ SD activity resulted from NDH-2 inhibition, we measured pMMO (% of control) activity in cells treated with 0, 50, or 500 ␮M DPI, lysed the 5 ␮M DPI, 5 mM NADH for 0 min 11.9 Ϯ 3.4 (17.2) cells, and isolated membrane fractions to measure NADH- 5 ␮M DPI, 5 mM NADH for 3 min 5.6 Ϯ 2.6 (8.1) dependent NDH-2 and pMMO activities in vitro (Table 2). 5 ␮M DPI, 5 mM NADH for 6 min 3.9 Ϯ 0.8 (5.6) ␮ Ϯ Most (83%) of the whole-cell pMMO activity remained after 5 M DPI, 5 mM NADH for 15 min 2.8 1.3 (4.0) ␮ 0 ␮M DPI, 5 mM NADH for 0 min 69.1 Ϯ 4.1 (100) treatment with 50 M DPI, whereas only 27% of NDH-2 activity was recovered in the membrane fraction, relative to an 0 ␮M PQ, 5 ␮M DPI 8.2 Ϯ 1.4 (11.7) untreated control (Table 2). The percentage of NADH-driven ␮ ␮ Ϯ 25 M PQ, 5 M DPI 22.1 2.8 (31.5) pMMO activity recovered in the membrane fraction (16%) was 50 ␮M PQ, 5 ␮M DPI 25.9 Ϯ 1.1 (37.0) ␮ ␮ Ϯ comparable to the recovered NDH-2 activity (27%), rather 100 M PQ, 5 M DPI 34.7 3.6 (49.5) Downloaded from 0 ␮M PQ, 0 ␮M DPI 70.1 Ϯ 5.7 (100)

a M. capsulatus membranes were incubated at 45°C for the indicated time in the presence of 5 mM NADH and 0 or 5 ␮M DPI before initiating the assay with propene. M. capsulatus membranes were incubated for 3 min at 45°C with DPI, PQ, and 1 ml of propene before initiating the assay with NADH. Values represent the means of three or four measurements. Numbers in parentheses are the activities expressed as a percentage of that of the 0 ␮M DPI control.

resulted in partial protection of pMMO activity from DPI http://jb.asm.org/ inhibition (i.e., 88% inhibition in the absence of PQ, relative to 50% inhibition in the presence of 100 ␮M PQ) (Table 1). According to our proposed electron transfer pathway (Fig. 1A) and the mechanism in Fig. 2, DPI is expected to inhibit pMMO turnover by inactivating NDH-2, thereby disrupting electron transfer from NADH to the monooxygenase. To further elucidate the role of DPI in blocking the electron transfer pathway (Fig. 1A), we examined its effect on hydroquinol- dependent pMMO activity. For this experiment we used de- on September 6, 2018 by guest tergent-solubilized pMMO, which is not active with NADH alone (40), but is active with exogenous hydroquinols or with NADH plus exogenous quinones and NDH-2 (13, 40). As expected, the NADH-dependent activity of detergent- soluble pMMO was very sensitive to DPI. Only Ϸ5 ␮M DPI was required to inhibit 50% of the activity (Fig. 5A), which was similar to the amount required to inhibit membrane-bound pMMO activity (Fig. 4). Unexpectedly, the pMMO activity

driven by exogenous decyl-plastoquinol (PQH2) was also inhibited by DPI (Fig. 5A). However, the PQH2-dependent pMMO activity was markedly less sensitive to DPI than the NADH-dependent activity. For example, 50% inhibition of the Ϸ ␮ PQH2-dependent activity required 70 M DPI, relative to Ϸ5 ␮M DPI for the NADH-dependent activity (Fig. 5A). To determine the effect of DPI on pMMO activity in whole cells, we treated M. capsulatus cells with DPI in the presence of formate, washed the cells thoroughly, then assayed pMMO FIG. 5. DPI inhibition proﬁles of hydroquinol- and NADH-driven activity with either formate, formaldehyde, or duroquinol as pMMO activities in vitro (A) and hydroquinol- formaldehyde-, and formate-driven pMMO activities in vivo (B). In A, the activity of the electron donor. DPI did inhibit whole-cell pMMO activity detergent-solubilized pMMO was measured in the presence of DPI ␮ in a concentration-dependent manner (Fig. 5B). Furthermore, with 5 mM NADH plus 350 MPQor3mMPQH2 as the reductant. the inhibition was independent of the electron donor; the same Activities are expressed as a percentage of the speciﬁc activity in the Ϫ1 Ϫ1 extent of inhibition is observed with formate, formaldehyde, absence of DPI (17.8 and 6.4 nmol min mg for NADH and PQH2-driven controls, respectively). (B) M. capsulatus cells were in- and duroquinol (Fig. 5B). The concentration of DPI required cubated for 5 min at 45°C with DPI and 5 mM formate, washed three for 50% inhibition of whole-cell pMMO activity was similar to times, and assayed for pMMO activity with either 5 mM formate, 5 that needed for inhibition of the hydroquinol-driven activity of mM formaldehyde, or 2 mM duroquinol as the reductant. Activities are expressed as a percentage of that of the 0 ␮M DPI control (205.4, detergent-solubilized pMMO (Fig. 5) and much higher than Ϫ1 Ϫ1 the concentration required to inhibit the NADH-dependent 225.1, and 130.8 nmol min mg for the formate-, formaldehyde-, and duroquinol-driven controls, respectively). Each data point is re- activities of pMMO and NDH-2 in vitro. For example, 50% ported as the mean value Ϯ standard deviation of three or four mea- inhibition of whole-cell pMMO activity required Ϸ70 ␮M DPI surements. VOL. 186, 2004 INHIBITION OF pMMO AND AMO BY DIPHENYLIODONIUM 933

TABLE 2. NDH-2 and pMMO activity after DPI treatment of M. capsulatus cellsa

Ϫ Ϫ Mean sp act (nmol min 1 mg 1) Ϯ SD (% of control) Incubation conditions Formate-dependent pMMO NADH-dependent pMMO NDH 0 ␮M DPI, 5 mM formate 185.9 Ϯ 8.4 (100) 201.5 Ϯ 21.9 (100) 583.8 Ϯ 84.1 (100) 50 ␮M DPI, 5 mM formate 154.3 Ϯ 27.1 (83) 32.4 Ϯ 3.2 (16.2) 158.1 Ϯ 48.7 (27.1) 500 ␮M DPI, 5 mM formate 52.6 Ϯ 18.1 (28.3) 4.3 Ϯ 1.2 (2.1) 24.6 Ϯ 6.3 (4.2) 50 ␮M DPI, no formate 155.6 Ϯ 12.7 (83.7) 152.3 Ϯ 11.0 (75.6) 517.7 Ϯ 34.3 (88.7)

a Cells were incubated for 10 min at 45°C under the conditions described and then washed three times with 50 mM PIPES (pH 7.2). A portion of each sample was saved for in vivo pMMO assays, and the remainder was lysed and the membranes were isolated and assayed for in vitro NADH-driven pMMO and NDH activities. Values reported are the mean of 8 to 14 measurements. Numbers in parentheses are the activities expressed as a percentage of that of the 0 ␮M DPI control.

than the whole-cell pMMO activity (83%) (Table 2). Similar quires turnover of AMO and is thereby prevented by anaerobic trends were observed for whole cells treated with 500 ␮M DPI; conditions or the presence of alternative substrates that com- Downloaded from 28% of whole-cell pMMO activity remained, whereas only 4% pete for the hydroxylation site of AMO (23, 26, 30). We there- and 2% of NDH-2 and pMMO activity, respectively, remained fore investigated whether the presence of hydrocarbon sub- in the membrane fraction (Table 2). strates or removal of oxygen could similarly protect AMO from To ensure that the effect of DPI on the membrane fraction DPI. Control samples were incubated in buffer alone, whereas was not enhanced by cell lysis fostering access of adventitiously bound DPI to its target sites, the cells were washed extensively prior to lysis. In addition, a control sample was prepared by incubating cells with 50 ␮M DPI in the absence of formate. http://jb.asm.org/ The NDH-2 and pMMO activities in membranes from this control were four- to ﬁvefold greater than in the sample treated with 50 ␮M DPI and formate and only slightly lower than that of the untreated control (Table 2). Thus, the differ- ence between whole-cell and membrane activities is not due to the presence of adventitious DPI during lysis.

DPI inhibition of AMO. DPI also inhibited NH3-dependent oxygen uptake and ethylene oxidation catalyzed by AMO in whole cells of N. europaea, with 50% inhibition observed at on September 6, 2018 by guest Ϸ100 ␮M DPI (Fig. 6). In these experiments, N. europaea cells were incubated with DPI and then washed thoroughly to remove unreacted DPI before enzyme activity was measured. The extensive washing did not restore AMO activity (Fig. 6),

suggesting that the inhibition was irreversible. NH3-dependent O2 uptake by AMO in cell-free lysates was completely inhibited by 50 ␮M DPI, but detailed characterization was prevented by the instability of AMO in vitro. As discussed above, ammonia oxidation in N. europaea is dependent on HAO to supply reducing equivalents from the

oxidation of hydroxylamine (3). Hydroxylamine-dependent O2 uptake was unaffected by DPI (Fig. 6A), indicating that HAO and the electron transfer pathway from HAO to the terminal oxidase were not disrupted by DPI. DPI also inhibited both hydrazine- and duroquinol-driven ethylene oxidation activities of AMO in whole cells (Fig. 6B). Furthermore, nearly identical inhibition proﬁles were observed for these two reductants, with Ϸ ␮ FIG. 6. Effect of DPI on HAO and AMO activities in whole cells of 50% inhibition at 200 M DPI (Fig. 6B). This is similar to N. europaea. Cells were incubated with DPI for 5 min at room tem- the concentration required for 50% inhibition of ammonia- perature, washed ﬁve times, and assayed for AMO and HAO activity. dependent O uptake (Fig. 6A), as well as pMMO activity in (A) AMO activity was measured as the rate of oxygen uptake in the 2 ϩ whole cells (Ϸ70 ␮M, Fig. 5B), and at least an order of mag- presence of 3.75 mM NH4 and is expressed as a percentage of the 0 ␮ ␮ Ϫ1 Ϫ1 nitude greater than required for inhibition of NADH-depen- M DPI control activity (1.05 mol min mg ). HAO activity was measured after adding 0.6 mM allylthiourea to inhibit AMO, followed dent activities of NDH-2 and pMMO in membrane fractions by 1.25 mM NH OH. Activity is expressed as a percentage of the 0 ␮M 2 Ϫ Ϫ (less than 5 ␮M, Fig. 4). DPI control (256 nmol min 1 mg 1). (B) AMO activity of the DPI- In view of the much broader substrate range of AMO rela- treated cells was measured as the rate of ethylene epoxidation with ␮ ␮ tive to pMMO (24, 27, 29), we considered the possibility that either 500 M hydrazine or 500 M duroquinol as the reductant. Activities are expressed as a percentage of the 0 ␮M DPI control Ϫ Ϫ hydroxylation of DPI caused the inactivation of AMO, as is the sample (348 and 165 nmol min 1 mg 1 for the hydrazine-driven and case for certain alkynes, cyclopropanes, and aniline derivatives duroquinol-driven controls, respectively). Each data point is reported (30). Inactivation by these mechanism-based inhibitors re- as the mean value Ϯ standard deviation of four measurements. 934 SHIEMKE ET AL. J. BACTERIOL.

TABLE 3. Protection of AMO by allylthiourea but not by DISCUSSION AMO substrates or anaerobic conditionsa Inactivation of NDH-2 by DPI and its effect on pMMO ac- Mean AMO activity Mean AMO activity Protectant with DPI only with DPI and protectant tivity. The substrate oxidation mechanism of pMMO and (% of control) Ϯ SD (% of control) Ϯ SD AMO remains poorly characterized due to their low and un- stable activity in vitro (27, 31, 51). The lack of robust activity Allylthiorrea (1 mM) 47.6 Ϯ 5.7 90.4 Ϯ 6.8 Methane (3 ml) 27.0 Ϯ 8.4 22.1 Ϯ 7.4 in vitro may arise from the use of nonphysiological reducing Ethane (3 ml) 27.2 Ϯ 6.2 23.2 Ϯ 5.1 agents that are unable to efﬁciently reduce the monooxygen- Ϯ Ϯ Phenol (1 mM) 23.3 3.9 26.5 4.9 ases. Hence, a better understanding of the physiological mech- Toluene (1 mM) 32.9 Ϯ 5 26.7 Ϯ 6.6 Anaerobic 36.8 Ϯ 7.1 19.2 Ϯ 9.3 anism for reduction of pMMO and AMO may lead to increased Hydrazineb (500 ␮M) 77.4 Ϯ 5.9 43.3 Ϯ 4.8 stability and activity in vitro and facilitate characterization of these novel enzymes. a For each protectant, three samples of N. europaea cells were prepared: one with 200 ␮M DPI, one with 200 ␮M DPI and the indicated protectant, and a In previous publications we proposed the electron transfer

control with no DPI and no protectant. These samples were incubated at room pathway to pMMO that is illustrated by the solid lines in Fig. Downloaded from temperature for 5 min, washed four times with 50 mM phosphate (pH 8), and assayed for AMO activity by measuring the rate of ethylene oxidation as de- 1A. This proposal was based on the observation that pMMO scribed in the text. Activities are given relative to the activity of the control activity could be driven by either exogenous hydroquinols or sample and represent the mean of three measurements. exogenous quinones plus NADH and NDH-2 under conditions b These samples contained 100 ␮M DPI rather than 200 ␮M as in all the other samples. in vitro where NADH alone is ineffective as a reductant (13, 40). This proposal could be tested by determining if inhibition of NDH-2 prevents turnover of pMMO. However, there are no experimental samples included either DPI or DPI plus the general inhibitors of NDH-2 enzymes; flavone inhibits some hydrocarbon substrate. All samples were washed extensively NDH-2 enzymes but is ineffective against the NDH-2 from http://jb.asm.org/ prior to measuring ethylene oxidation activity of AMO. M. capsulatus (13). The AMO substrates methane, ethylene, toluene, and phe- In the present study we demonstrated that DPI is a potent, Ͼ nol had no protective effect; nearly identical levels of AMO irreversible inhibitor of purified M. capsulatus NDH-2: 97% ␮ activity were recovered after incubation with DPI regardless of inhibition was observed in the presence of 10 M DPI (Fig. 3, the presence of these hydrocarbons (Table 3). Incubation of N. inset). DPI also irreversibly inhibited NDH-2 activity in iso- europaea cells with DPI under anaerobic conditions also failed lated membrane fractions. This inhibition required reduction to protect AMO from inhibition. In fact, removal of oxygen of NDH-2, since DPI had little effect on enzyme activity in the resulted in recovery of Ϸ50% less AMO activity relative to absence of NADH (Fig. 3). Thus, inhibition of NDH-2 by DPI incubation in the presence of oxygen (Table 3). The increased has the characteristics one would expect from its typical mech- on September 6, 2018 by guest sensitivity to DPI under anaerobic conditions could be due to anism of action on flavoproteins (Fig. 2) (10, 35, 43), in that inhibition was irreversible and required reduction by NADH enhanced reduction of the DPI binding site without O2 to serve as the terminal electron acceptor. This explanation is consis- (Fig. 3 and 4). We conclude that DPI reacts only with the tent with similar enhancement of DPI inhibition observed in reduced form of NDH-2, resulting in covalent modification of the presence of the electron donor hydrazine (Table 3), sug- its flavin cofactor and inactivation of its normal redox cycle. gesting that reduction is a precursor to DPI binding, as ex- Having shown that DPI will inactivate NDH-2, we investi- pected from its proposed mechanism of inactivation (Fig. 2). gated whether NADH-coupled pMMO activity is also inhib- The only agent found to protect AMO from DPI inhibition ited by DPI. We found that titration of membrane fractions was allylthiourea, which inhibits AMO by chelating copper with DPI in the presence of NADH resulted in similar inhibi- (21). A similar chelator, thiourea, is known to protect AMO tion profiles for NADH-dependent pMMO and NDH-2 activ- from mechanism-based inactivation by alkynes, anilines, and ities (Fig. 4). Furthermore, the inhibitory effect of DPI on cyclopropanes (30). We found that 90% of AMO activity was pMMO activity was irreversible and dependent on the pres- recovered after incubation with 200 ␮M DPI in the presence of ence of NADH, consistent with inactivation of NDH-2 as the 1 mM allylthiourea, whereas only 48% remained after DPI cause of pMMO inhibition. Protection of pMMO activity by treatment in the absence of allylthiourea (Table 3). In this PQ (Table 1) is also consistent with inhibition caused by inac- experiment, two control samples were prepared, with no allyl- tivation of NDH-2. PQ is an efficient electron acceptor for thiourea and no DPI or with 1 mM allylthiourea and no DPI. NDH-2 (13), so increased concentrations of PQ would de- The experimental samples contained no allylthiourea and 200 crease the steady-state concentration of reduced NDH-2, pro- ␮M DPI or 1 mM allylthiourea and 200 ␮M DPI. The activities viding less opportunity for reaction with DPI and therefore less reported in Table 3 for samples with DPI alone are relative to disruption of electron flow to pMMO. A similar result was the control with no DPI or allylthiourea. Similarly, the activi- observed for DPI inhibition of cytochrome P450 reductase ties of the samples incubated with both allylthiourea and DPI (43), whereby increased concentrations of the electron accep- are reported relative to the control treated with allylthiourea tor (cytochrome c) resulted in decreased sensitivity to DPI. We alone (Table 3). The buffer used to wash these samples con- therefore conclude that electron transfer from NADH to ␮ tained 50 M CuSO4 to reconstitute AMO activity lost due to pMMO in vitro requires NDH-2 and that DPI inhibits pMMO copper chelation by allylthiourea. In this way we determined by blocking this pathway (Fig. 1A). that the loss of activity due to incubation with allylthiourea DPI inhibition of pMMO and AMO that is independent of alone is almost fully reversible. The allylthiourea-treated sam- NDH-2. We have proposed that NADH, NDH-2, and the qui- ple retained 93% of the AMO activity in an untreated control. none pool convey reducing equivalents from formaldehyde and VOL. 186, 2004 INHIBITION OF pMMO AND AMO BY DIPHENYLIODONIUM 935 formate to pMMO in vivo (solid lines in Fig. 1A) (13, 40). On is located downstream of the quinone pool. The PQQ-contain- this basis, we expected DPI to inhibit formate- or formalde- ing dehydrogenase is not likely to be the DPI-binding site, hyde-driven pMMO activity in whole cells at a concentration since it is specific for formaldehyde (50) and therefore could similar to that observed for inhibition of NADH-driven activity not be responsible for inhibition of formate- or duroquinol- in vitro (Ϸ10 ␮M). We found that formate- and formaldehyde- driven pMMO activity. Similarly, binding of DPI to the cyto- driven pMMO activities were both inhibited by DPI (and with chrome bc1 complex could account for inhibition of pMMO identical sensitivity), but these activities were less sensitive only if it mediates electron transfer from formate, formalde- than NADH-driven in vitro activity by at least an order of hyde, and duroquinol. This seems unlikely, since several labo- magnitude (cf. Fig. 4 and 5B). The obstacle of crossing the cell ratories have reported that duroquinol supports the activity of membrane might account for some of the reduced sensitivity of pure pMMO in the absence of any cytochrome bc1 (4, 11, 31, pMMO in whole cells to DPI. However, treatment of whole 51). cells with 50 ␮M DPI caused inactivation of 73% of the It was recently reported that maximal activity of purified NDH-2 activity but only 17% of the pMMO activity (Table 2), pMMO was observed with NADH, NDH-2, and duroquinol indicating that DPI can readily interact with and substantially (11), suggesting that no other proteins are required to mediate Downloaded from inhibit NDH-2 in whole cells without significantly affecting electron transfer from hydroquinols to pMMO. Consistent pMMO activity. with this proposal is DPI inhibition of the PQH2-driven activity It is possible that the turnover rate of NDH-2 is much of detergent-solubilized pMMO (Fig. 5A). Significantly, the Ϸ greater than that of pMMO in vivo, such that NDH-2 activity PQH2-driven activity is 15-fold less sensitive to DPI than the remaining after DPI treatment was sufficient to support the NADH-driven activity (Fig. 5). Given their similar response to relatively high level of whole-cell pMMO activity observed DPI, we propose that a single DPI-binding site is responsible (Table 2). However, if the residual NDH-2 activity is sufficient for inhibiting formaldehyde-, formate-, and duroquinol-driven to support significant pMMO activity in vivo, it should support pMMO activity in vivo as well as plastoquinol-driven activity in http://jb.asm.org/ similar levels of pMMO activity in vitro. In fact, the NADH- vitro. This implies that the NDH-2-independent pathway in- driven pMMO activity recovered from DPI-treated cells was cludes the quinone pool and that binding of DPI to this rela- Ϸ5-fold lower than pMMO activity in vivo (Table 2). Further- tively low-affinity site blocks electron transfer from hydroquin- more, the percentage of NADH-driven pMMO activity was ols to pMMO (Fig. 1A), perhaps within the pMMO complex comparable to the percentage of recovered NDH-2 activity itself (see below). In contrast, NDH-2 activity is essential for rather than the whole-cell pMMO activity (Table 2). Thus, it NADH-driven pMMO activity, and the relatively greater sen- appears that electron transfer from formate to pMMO can sitivity of this activity is due to the high affinity of NDH-2 for substantially continue in the presence of DPI concentrations DPI (Fig. 3). sufficient to inactivate NDH-2. This result suggests the exis- DPI also inhibited AMO activity in cells of N. europaea (Fig. on September 6, 2018 by guest tence of an additional electron transfer pathway from formate 6). This inhibition appeared to be irreversible, since extensive to pMMO that is independent of NDH-2 but also contains a washing of the cells after incubation with DPI did not restore DPI-sensitive redox site (dashed lines in Fig. 1A). This second activity. Mechanism-based inhibition resulting from hydroxy- DPI-binding site has a lower affinity for the inhibitor than lation of DPI at the AMO active site does not appear to be NDH-2, since Ϸ15-fold more DPI is required to inhibit for- responsible for the effect of DPI on AMO activity (Table 3). In mate-driven pMMO activity relative to NDH-2 activity (Fig. addition, it is unlikely that inactivation of an NDH-2 enzyme by 5B and Table 2). DPI is responsible for inhibition of AMO activity, since the The redox cofactors and proteins that constitute this addi- electron transfer pathway from HAO to AMO in N. europaea tional electron transfer pathway from formate to pMMO re- is thought to be independent of NADH (the redox potential of Ϫ Ϸ main unknown at present. Both pathways illustrated in Fig. 1A the NH2OH/NO2 couple is 300 mV higher than that of ϩ appear to operate simultaneously in vivo, since treatment of NADH/NAD ) (8, 48). However, it is possible that an enzyme whole cells with formate and DPI results in inhibition of NDH- similar to NDH-2 mediates reduction of the quinone pool (and 2-dependent as well as NDH-2-independent pathways (Table ultimately AMO) in N. europaea, with an electron donor other 2). The NDH-2-independent pathway could consist of a single than NADH (a reduced cytochrome, for example). A search of DPI-sensitive protein that transfers electrons from formate the N. europaea genome revealed a single gene with sequence directly to pMMO. A pyrroloquinoline quinone (PQQ)-con- homology (26% identity, 41% similarity) to the NDH-2 of taining formaldehyde dehydrogenase was recently isolated Escherichia coli (7), but a knockout mutation of this gene had from M. capsulatus membranes that appears to transfer reduc- no effect on AMO activity or inhibition by DPI (data not ing equivalents specifically from formaldehyde to the cyto- shown). We propose instead that DPI disrupts electron trans- chrome bc1 complex (50). It was suggested that the reduced fer from the quinone pool to AMO in a manner analogous to cytochrome bc1 complex subsequently transfers these reducing that suggested above for the NDH-2-independent inhibition of equivalents to pMMO (50). This proposed electron transfer pMMO (Fig. 1B). chain could be responsible for the NDH-2 independent DPI DPI appears to specifically inhibit AMO activity, having no inhibition of pMMO, since both PQQ and low-potential b- effect on hydroxylamine-dependent oxygen uptake by N. euro- hemes are reportedly sensitive to DPI (6, 16). However, the paea cells (Fig. 6A). Therefore, neither HAO turnover nor the response of whole-cell pMMO activity to DPI was independent electron transfer pathway from HAO to the terminal oxidase is of the source of reducing equivalents (Fig. 5B), suggesting that affected by DPI. If we assume that the quinone pool is a a single DPI-binding site blocks electron transfer to pMMO common source of reducing equivalents for both AMO and the from formate, formaldehyde, and duroquinol, and that this site terminal oxidase (as suggested by Hooper and coworkers [48] 936 SHIEMKE ET AL. J. BACTERIOL. and illustrated in Fig. 1B), then the specificity of DPI inhibition membrane bound NADH dehydrogenase of Escherichia coli (NDH-2) and analysis of its interaction with ubiquinone analogues. FEBS Lett. 467:105– of AMO is consistent with disruption of electron flow from 110. reduced quinones to the monooxygenase. Furthermore, iden- 8. Bock, E., H.-P. Koops, H. Harms, and B. Ahlers. 1991. The biochemistry of tical levels of AMO inhibition by DPI were observed with nitrifying organisms, p. 171–200. In J. M. Shively and L. L. Barton (ed.), Variations in autotrophic life. Academic Press Limited, San Diego, Calif. either hydrazine or duroquinol as the reductant (Fig. 6B). 9. Burrows, K. J., A. Cornish, D. Scott, and I. J. Higgins. 1984. Substrate HAO and the quinone pool are intermediates when hydrazine specificities of the soluble and particulate methane monooxygenase of donates electrons to AMO and the terminal oxidase (3), where- Methylosinus trichosporium OB3b. J. Gen. Microbiol. 130:3327–3333. 10. Chakraborty, S., and V. Massey. 2002. Reaction of reduced flavins and as duroquinol is thought to directly reduce AMO (or perhaps flavoproteins with diphenyliodonium chloride. J. Biol. Chem. 277:41507– the endogenous quinone pool) (39, 48). DPI could disrupt 41516. electron transfer to AMO from hydrazine and duroquinol by 11. Choi, D. W., R. C. Kunz, E. S. Boyd, J. D. Semrau, W. E. Antholine, J. I. Han, J. A. Zahn, J. M. Boyd, A. M. de la Mora, and A. A. DiSpirito. 2003. The reacting with redox components unique to each reductant. membrane-associated methane monooxygenase (pMMO) and pMMO- However, the identical sensitivity to DPI is consistent with NADH:quinone oxidoreductase complex from Methylococcus capsulatus 185: disruption of electron flow at a point common to both reduc- Bath. J. Bacteriol. 5755–5764.

12. Colby, J., D. I. Stirling, and H. Dalton. 1977. The soluble methane mono- Downloaded from tants, i.e., between the quinone pool and AMO (Fig. 1B). oxygenase of Methylococcus capsulatus (Bath). Its ability to oxygenate n- The precise identity of the DPI- binding site in N. europaea alkanes, n-alkenes, ethers, and alicyclic, aromatic and heterocyclic compounds. Biochem. J. 165:395–402. (and by analogy the low-affinity DPI-binding site in M. capsu- 13. Cook, S. A., and A. K. Shiemke. 2002. Evidence that a type-2 NADH:quinone latus) remains unknown. However, if hydroquinols react direct- oxidoreductase mediates electron transfer to particulate methane monoox- ly with AMO and pMMO, as has been proposed (13, 40, 48), ygenase in Methylococcus capsulatus. Arch. Biochem. Biophys. 398:32–40. 14. Cook, S. A., and A. K. Shiemke. 1996. Evidence that copper is a required then DPI must inhibit electron transfer between the hydro- cofactor for the membrane-bound form of methane monooxygenase. J. In- quinol-binding site and the hydroxylation site, i.e., within org. Biochem. 63:273–284. 15. Coves, J., C. Lebrun, G. Gervasi, P. Dalbon, and M. Fontecave. 1999. Over- the monooxygenase itself. Although there is no evidence for expression of the FAD-binding domain of the sulphite reductase flavopro- a flavin-binding site in either AMO or pMMO, DPI has been tein component from Escherichia coli and its inhibition by iodonium diphenyl http://jb.asm.org/ reported to react with redox cofactors other than flavins (6, chloride. Biochem. J. 342:465–472. 16. Doussiere, J., J. Gaillard, and P. V. Vignais. 1999. The heme component of 16). Protection of AMO by the copper chelator allylthiourea the neutrophil NADPH oxidase complex is a target for aryliodonium com- (Table 3) could be significant in this regard. Specific chelation pounds. Biochemistry 38:3694–3703. of copper has long been known to completely inhibit AMO (5, 17. Ensign, S. A., M. R. Hyman, and D. J. Arp. 1993. In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper. J. Bacte- 21) and pMMO (14, 42) in whole cells and membrane frac- riol. 175:1971–1980. tions, but the precise role and location of the labile copper was 18. Hamamura, N., C. M. Yeager, and D. J. Arp. 2001. Two distinct monooxygenases for alkane oxidation in Nocardioides sp. strain CF8. Appl. Environ. unknown. More recently, several laboratories have reported Microbiol. 67:4992–4998. that purified pMMO is also inhibited by chelation of copper (4, 19. Hanson, R. S., and T. E. Hanson. 1996. Methanotrophic bacteria. Microbiol. 31, 51), suggesting that this labile copper is an obligatory co- Rev. 60:439–471. on September 6, 2018 by guest 20. Holmes, A. J., A. Costello, M. E. Lidstrom, and J. C. Murrell. 1995. Evidence factor of the catalytic complex of pMMO and, by homology, that particulate methane monooxygenase and ammonia monooxygenase may AMO as well. Since removal of this labile copper protects be evolutionarily related. FEMS Microbiol. Lett. 132:203–208. AMO from inhibition by DPI, we tentatively suggest that it is 21. Hooper, A. B., and K. R. Terry. 1973. Specific inhibitors of ammonia oxidation in Nitrosomonas. J. Bacteriol. 115:480–485. required for electron transfer from the physiological electron 22. Hooper, A. B., T. Vannelli, D. J. Bergmann, and D. M. Arciero. 1997. donor (presumably hydroquinol) to the hydroxylation site. Fu- Enzymology of the oxidation of ammonia to nitrite by bacteria. Antonie Van ture investigations into the mechanism of DPI inhibition of Leeuwenhoek 71:59–67. 23. Hyman, M. R., and D. J. Arp. 1988. Acetylene inhibition of metalloenzymes. AMO and pMMO could provide significant information re- Anal. Biochem. 173:207–220. garding the redox chemistry of these novel monooxygenases. 24. Hyman, M. R., I. B. Murton, and D. J. Arp. 1988. Interaction of ammonia monooxygenase from Nitrosomonas europaea with alkanes, alkenes, and alkynes. Appl. Environ. Microbiol. 54:3187–3190. ACKNOWLEDGMENTS 25. Hyman, M. R., and P. M. Wood. 1984. Ethylene oxidation by Nitrosomonas europaea. Arch. Microbiol. 137:155–158. This work was supported by grant DE-FG03-97ER20266 from the 26. Hyman, M. R., and P. M. Wood. 1985. Suicidal inactivation and labelling of U.S. Department of Energy (to D.J.A.). Sabbatical support was pro- ammonia mono-oxygenase by acetylene. Biochem. J. 227:719–725. vided to A.K.S. from the West Virginia University Faculty Senate and 27. Juliette, L. Y., M. R. Hyman, and D. J. Arp. 1993. Mechanism-based inac- the office of the Office of the Associate Dean for Research of the tivation of of ammonia monooxygenase in Nitrosomonas europaea by allyl- WVU School of Medicine. sulfide. Appl. Environ. Microbiol. 59:3728–3735. 28. Juliette, L. Y., M. R. Hyman, and D. J. Arp. 1995. Roles of bovine serum albumin and copper in the assay and stability of ammonia monooxygenase REFERENCES activity in vitro. J. Bacteriol. 177:4908–4913. 1. Anthony, C. 1986. Bacterial oxidation of methane and methanol. Adv. Mi- 29. Keener, W. K., and D. J. Arp. 1994. Transformations of aromatic compounds crob. Physiol. 27:113–210. by Nitrosomonas europaea. Appl. Environ. Microbiol. 60:1914–1920. 2. Anthony, C. 1982. The biochemistry of methanotrophs. Academic Press, 30. Keener, W. K., S. A. Russell, and D. J. Arp. 1998. Kinetic characterization of London, England. the inactivation of ammonia monooxygenase in Nitrosomonas europaea by 3. Arp, D. J., L. A. Sayavedra-Soto, and N. G. Hommes. 2002. Molecular alkyne, aniline and cyclopropane derivatives. Biochim. Biophys. Acta 1388: biology and biochemistry of ammonia oxidation by Nitrosomonas europaea. 373–385. Arch. Microbiol. 178:250–255. 31. Lieberman, R. L., D. B. Shrestha, P. E. Doan, B. M. Hoffman, T. L. 4. Basu, P., B. Katterle, K. K. Andersson, and H. Dalton. 2003. The membrane- Stemmler, and A. C. Rosenzweig. 2003. Purified particulate methane mono- associated form of methane mono-oxygenase from Methylococcus capsulatus oxygenase from Methylococcus capsulatus (Bath) is a dimer with both mono- (Bath) is a copper/iron protein. Biochem. J. 369:417–427. nuclear copper and a copper-containing cluster. Proc. Natl. Acad. Sci. 100: 5. Bedard, C., and R. Knowles. 1989. Physiology, biochemistry, and specific 3820–3825. ϩ inhibitors of CH4,NH4 , and CO oxidation by methanotrophs and nitrifiers. 32. Lipscomb, J. D. 1994. Biochemistry of the soluble methane monooxygenase. Microbiol. Rev. 53:68–84. Annu. Rev. Microbiol. 48:371–399. 6. Bishop, A., M. A. Paz, P. M. Gallop, and M. L. Karnovsky. 1995. Inhibition 33. Murrell, J. C., I. R. McDonald, and B. Gilbert. 2000. Regulation of expres- of redox cycling of methoxatin (PQQ), and of superoxide release by phago- sion of methane monooxygenases by copper ions. Trends Microbiol. 8:221– cytic white cells. Free Radic. Biol. Med. 18:617–620. 225. 7. Bjorklof, K., V. Zickermann, and M. Finel. 2000. Purification of the 45 kDa, 34. Nguyen, H. H., S. J. Elliott, J. H. Yip, and S. I. Chan. 1998. The particulate VOL. 186, 2004 INHIBITION OF pMMO AND AMO BY DIPHENYLIODONIUM 937

methane monooxygenase from Methylococcus capsulatus (Bath) is a novel 43. Tew, D. G. 1993. Inhibition of cytochrome P450 reductase by the diphenyli- copper-containing three-subunit enzyme. Isolation and characterization. odonium cation. Kinetic analysis and covalent modifications. Biochemistry J. Biol. Chem. 273:7957–7966. 32:10209–10215. 35. O’Donnell, B. V., D. G. Tew, O. T. Jones, and P. J. England. 1993. Studies on 44. Truesdale, G. A., and A. L. Downing. 1954. Solubility of oxygen in water. the inhibitory mechanism of iodonium compounds with special reference to Nature 173:1236. neutrophil NADPH oxidase. Biochem. J. 290:41–49. 45. Tsien, H. C., G. A. Brusseau, R. S. Hanson, and L. P. Wackett. 1989. 36. O’Donnell, V. B., G. C. Smith, and O. T. Jones. 1994. Involvement of phenyl Biodegradation of trichloroethylene by Methylosinus trichosporium OB3b. radicals in iodonium inhibition of flavoenzymes. Mol. Pharmacol. 46:778– Appl. Environ. Microbiol. 55:3155–3161. 785. 46. Wackett, L. P., G. A. Brusseau, S. R. Householder, and R. S. Hanson. 1989. 37. Prior, S. D., and H. Dalton. 1985. Acetylene as a suicide substrate and active Survey of microbial oxygenases: trichloroethylene degradation by propane- site probe for methane monooxygenase from Methylococcus capsulatus oxidizing bacteria. Appl. Environ. Microbiol. 55:2960–2964. (Bath). FEMS Microbiol. Lett. 29:105–109. 47. Wallar, B. J., and J. D. Lipscomb. 1996. Dioxygen activation by enzymes 38. Rasche, M. E., R. E. Hicks, M. R. Hyman, and D. J. Arp. 1990. Oxidation of containing binuclear non-heme iron clusters. Chem. Rev. 96:2625–2658. monohalogenated ethanes and n-chlorinated alkanes by whole cells of Ni- Whittaker, M., D. Bergmann, D. Arciero, and A. B. Hooper. trosomonas europaea. J. Bacteriol. 172:5368–5373. 48. 2000. Electron 39. Shears, J. H., and P. M. Wood. 1986. Tri- and tetramethylhydroquinone as transfer during the oxidation of ammonia by the chemolithotrophic bacte- electron donors for ammonia monooxygenase in whole cells. FEMS Micro- rium Nitrosomonas europaea. Biochim. Biophys. Acta 1459:346–355. biol. Lett. 33:281–284. 49. Yu, S. S., K. H. Chen, M. Y. Tseng, Y. S. Wang, C. F. Tseng, Y. J. Chen, D. S. Downloaded from 40. Shiemke, A. K., S. A. Cook, T. Miley, and P. Singleton. 1995. Detergent Huang, and S. I. Chan. 2003. Production of high-quality particulate methane solubilization of membrane-bound methane monooxygenase requires plas- monooxygenase in high yields from Methylococcus capsulatus (Bath) with a toquinol analogs as electron donors. Arch. Biochem. Biophys. 321:421–428. hollow-fiber membrane bioreactor. J. Bacteriol. 185:5915–5924. 41. Smith, D. D., and H. Dalton. 1989. Solubilisation of methane monooxygen- 50. Zahn, J. A., D. J. Bergmann, J. M. Boyd, R. C. Kunz, and A. A. DiSpirito. ase from Methylococcus capsulatus (Bath). Eur. J. Biochem. 182:667–671. 2001. Membrane-associated quinoprotein formaldehyde dehydrogenase 42. Stanley, S. H., S. D. Prior, D. J. Leak, and H. Dalton. 1983. Copper stress from Methylococcus capsulatus Bath. J. Bacteriol. 183:6832–6840. underlies the fundamental change in intracellular location of methane 51. Zahn, J. A., and A. A. DiSpirito. 1996. Membrane-associated methane mono- monooxygenase in methane oxidizing organisms. Biotechnol. Lett. 5:487– oxygenase from Methylococcus capsulatus (Bath). J. Bacteriol. 178:1018– 492. 1029. http://jb.asm.org/ on September 6, 2018 by guest