J. theor. Biol. (1976) 58, 325--335
A Possible Role of Free Radicals in the Oxidation of Methane by Methylococcus capsulatus D. W. HtrrcmNsoN'l"
Department of Molecular Sciences, University of Warwick, Coventry CV4 7AL, England AND
R. WHITrENBURY AND S . DALTON
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, England (Received 30 May 1975) A mechanism involving free radicals is proposed for the oxidation of methane by Methylococcus capsulatus. Removal of a hydrogen atom from methane can be effected by oxygen in the form of a hydroperoxide-iron complex. Subsequent steps in the oxidation of the methyl radical so formed to formaldehyde can be accounted for by known free radical reactions. The oxidation of substrates other than methane (e.g. dimethyl ether, carbon monoxide or ammonia) can be accommodated by variations in the initial free radical mechanism. 1. Introduction
Methane oxidizing bacteria are Gram-negative non-endosporing aerobes which use only C1 compounds, as combined sources of carbon and energy (in the absence of other carbon compounds). It has been tentatively assumed that oxidation of methane is initiated by a mixed-function oxidase (or hydroxylase as it is now known) mechanism similar to that which operates within other n-alkane (C2 +) oxidizing microbes. Indirect evidence for a typical hydroxylase mechanism was provided by (a) the observed incorporation of 1SOz into cell material (Leadbetter & Foster, 1959; Bryan-Jones & Wilkinson, 1971) and into methanol accumulated during methane oxidation (Higgins & Quayle, 1970); and (b). the stimulation by NADH of oxygen consumption by cell-free extracts in th e presence of methane or carbon monoxide (Ribbons & Michalover, 1970; Ferenci, 1974). So far, however, stoichiometric evidence which would support unambiguously a hydroxylase mechanism in cell-free t Addressfor reprint requests. 325
326
D.W.
HUTCHINSON,
R. WHITTENBURY
AND
H. DALTON
extracts of methane oxidisers, identical to that in microbes oxidising C2+ hydrocarbons, is missing. A major problem with cell-free work in this field has been the extreme lability of activity which is limited to 24 hr following preparation. To the experimental evidence above can be added circumstantial evidence to suggest that a conventional hydroxylase function may not be present at all The following observations raise this possibility: (I) Dimethyl ether is excreted and can be utilized as the sole carbon and energy source. Dimethyl carbonate can also be utilized (BryanJones & Wilkinson, 1971). (2) Carbon monoxide and ammonia are oxidized apparently by the same "oxygenase" activity responsible for methane oxidation (Hubley, Mitton & Wilkinson, 1974; Colby, Dalton & Whittenbury, 1975). (3) Other hydrocarbons (e.g. ethane or propane) are oxidized but not used as sole carbon and energy sources. Microbes oxidizing and growing upon n-alkanes (C2+) conversely do not oxidize methane. (4) Methanol, although utilized, is toxic in the medium in concentration as low as 0.01 ~o (v/v). Tolerant strains can be obtained but these are mutants which have very low oxidation rates compared with the wild type (Reed, I975). (5) The molar growth yields obtained with methane are either higher or similar to molar growth yields obtained with methanol. If a mixed function oxidase were involved, oxidation of NADH during the step concerned with the incorporation of oxygen would have to be coupled to ATP synthesis to account for the observed growth yields. To date no activity of this sort has been reported for known hydroxylases. Further consideration of methane oxidation involving sources of NADH for the oxidation step and carbon for growth reveal problems of accounting for growth yields observed, particularly in the Type 1 group of methane oxidisers. Based on the assumption that methane is initially oxidized by a hydroxylase mechanism, Van Dijken & Harder (1975) have concluded that it is necessary to postulate a reverse electron transport system to generate sufficient NADH for cell growth. Under such circumstances, the maximum calculated yield would be 0.91 g cells/g methane. In the absence of an hydroxylase mechanism (and hence a system that requires no input of energy or reductant), the maximum yield possible would be 1.46 g cells/g methane. Since yield values for Methylococcus have been reported to be higher than those calculated for an hydroxylase mechani~sm [e.g. 1.1 (Whittenbury, Phillips & Wilkinson, 1970), 1-0 (Harwood & Pirt, 1972) amongst others of the same order], then the existence of such a mechanism must be in
METHANE OXIDATION
BY M . C A P S U L A T U S
327
some doubt. In addition, since the yield coefficient of cells fixing nitrogen is only 5 7o less than those assimilating nitrate (Dalton & Whittenbury, 1975) and since nitrogen fixation represents a drain on the reducing equivalents of the cells, we find it difficult to rationalize this with the concept of an hydroxylase mechanism in which the cell has to expend energy to produce sufficient NADH for methane assimilation. Furthermore, Van Dijken & Harder have made a number of assumptions, e.g. YArP = 10"5, P/O ratios from NADH = 3 which are acceptable in a theoretical treatment but which need to be justified experimentally. However, one assumption made by these authors which is misleading and which we find difficult to accept is that there is no ATP production resulting from the oxidation of NADH if a reverse electron transport (and hence an hydroxylating) mechanism is functioning. To support this contention Van Dijken & Harder point out that Ribbons, Harrison & Wadzinski (1970) found very low levels of NADH oxidase activity in Methylococcus extracts. In fact, the values for NADH oxidase activity quoted by both Ribbons & Michaelover (1970) and Ferenci (1974) for methane oxidizing extracts were about 40 nmol NADH oxidized/ min/mg protein. Such a value is to be expected for an aerobe that relies extensively on NADH oxidation to obtain its energy for growth. This evidence would suggest that no reverse electron transport operates in methane-oxidizing bacteria and hence that there is no mixed function oxidase. We consider that an alternative to the hydroxylase mechanism for methane oxidation needs to be considered and that a system involving free radicals could possibly account for all the anomalies we have outlined. I-Iydroxylases have been studied for a number of years and enzyme systems which can hydroxylate comparatively unreactive molecules have been detected in both animals and bacteria (Hayaishi, 1962, 1969; Ullrich, 1972). The oxidation of methane possibly via methanol to formaldehyde by methylobacteria has not been studied in such detail, although the first stage in this oxidation can be regarded as similar to hydroxylation by a hydroxylase. A major problem in the oxidation of hydrocarbons and other unreactive molecules by molecular oxygen is the activation of the oxygen so that it will react with C - - H bonds. In some cases it appears that cytochrome P-450 is involved and although the mechanism of activation by this cytochrome is unclear, peroxide and superoxide ions have also been suggested as possible intermediates (Ullrich, 1972). Recent studies on the co-oxidation by Methylococcus capsulatus of substrates in the presence of methane coupled with the circumstantial evidence prompt us to propose a free radical mechanism for the first stages in the oxidation of methane and other substrates by this organism. The proposal of a free radical mechanism is not novel. The appearance
328
D.W.
HUTCHINSON,
R. WHITTENBURY
AND
H. DALTON
CH 4
I
-H"
CHff
o2
C2H4 CH. tO2 . . . .
CH30~
•- ~ ' C H 2 - - C H 2 +
CH30
\ oI / I
I I
CH30 ~ CH5
HOCHEH~H
CH 3" CHsOCH 3 .t i I
I-H* I I
-CH 3"
c.~ocH;
-CH~ 11
-CO2/// /
/
/
c.3~coc. ~ I
"I
CH20
I-H" II CH3OCOOCH3
H20
2e
.coo. I NAD+
~NADH CO2
FIG. I. Possible p a t h w a y s involved in the free radical oxidation o f m e t h a n e . main pathways; ..... , ancillary pathways.
METHANE OXIDATION BY M. CAPSrILATeS
329
of methyl ketones during co-oxidative metabolism of higher alkanes by Pseudomonas methanica prompted the proposal of a free radical mechanism involving alkyl hydroperoxides (Leadbetter & Foster, 1960). Methyl ketones, it was suggested, were formed if the initial oxidative step led to a secondary alcohol and fatty acids were formed if the initial attack produced a primary alcohol. This scheme has been criticized by Markovetz (1971) on the grounds that if the secondary alkyl hydroperoxide is a transitory intermediate then the secondary alcohol need not be formed since the ketone could be formed directly from the hydroperoxide and the alcohol could be the product of reduction of the methyl ketone. However, Markovetz does suggest that a non-specific free radical mechanism could explain the appearance of subterminal oxidation products during the oxidation ofn-decane by P. aeruginosa. Neither scheme mentions the possibility that a free radical mechanism could function in the oxidation of methane. In view of the extreme reactivity of many of the free radical intermediates which might be involved, we propose that some free radicals may be stabilized by co-ordination to a metal and that some reactions may take place between reactants co-ordinated to the same or closely neighbouring metal ions. Methylococcus capsulatus contains both haem and non-haem iron as well as copper, and we propose that molecular oxygen co-ordinated to a metal (e.g. Fe ~I) may be sufficiently activated in the form of an iron(III) hydroperoxide to remove a hydrogen atom from methane. The oxidation of ferrous ions by molecular oxygen has been proposed to take place by hydrogen-atom transfer (George, 1954). This process is catalysed by cupric ions and it may be significant that metal chelating agents which are specific for copper inhibit methane oxidation by M. capsulatus (Wilkinson, 1974). Co-ordination of the ferrous iron with a suitable ligand, e.g. a cytochrome or sulphur-containing protein, may change the energy levels of the iron orbitals and hence assist the hydrogen-atom transfer reaction. For example, cytochrome P-450 is involved in the hydroxylation of camphor by P. putida and electron spin resonance studies indicate that the number of unpaired electrons in the iron atom of cytochrome P-450 increases when camphor is added to the hydroxylating system (Peterson, 1971). Removal of a hydrogen atom from methane by the iron(III) hydroperoxide would generate a methyl radical which could be stabilized by co-ordination to an iron atom. The iron-methyl could then react with molecular oxygen to give a methylperoxy iron derivative, this insertion reaction is known to occur with other metal alkyls (Sosnovsky & Brown, 1966). In addition to the iron-methyl, hydrogen peroxide is formed and this could be broken down homolytically, possibly by a metalloenzyme, to hydroxyl radicals. The latter could also participate in the removal of a
330
D. W . H U T C H I N S O N , R. W H I T T E N B U R Y AND H. D A L T O N
0"~0 02
•
OOH FeE°H2
"
+
WhereYepresents ligonded iron
LFe OHJ
1 CH4
02
+ H2 0 2
hydrogen atom from methane. Dissociation of the methylperoxy iron, followed by dimerization and fission of the dimer to molecular oxygen methoxy radicals (Howard, 1973) would then be the next process to occur. Abstraction of a hydrogen atom from the solvent by the methoxy radical would lead to methanol, alternatively loss of a hydrogen atom from the methoxy radical would generate formaldehyde, which could be oxidized further to formate and finally carbon dioxide by enzymes which do not involve free radicals (Patel & Hoare, 1971).
OOCH3 +
CH 00"
CuII/CuIT c%oo"
CH O0 i
OOCH3
CHsOH-t-'OH
CH20
2 CH30"+02
Although methanol is oxidized by M. capsulatus it is t o x i c at relatively low concentrations (Whittenbury, Phillips & Wilkinson, 1970). It could be
METHANE OXIDATION
331
BY M'. C A P S U L A T U S
argued from the free radical mechanism outlined above that methanol is not on the main path of methane oxidation in this organism but is formed by reduction of formaldehyde, the latter being the essential source of carbon for synthesis. If this were the case, then during the oxidation of methane with 180-labelled oxygen, some of the 180 might be lost from the formaldehyde by exchange with water. While the incorporation of 180 from oxygen into methanol has not been studied in M. capsulatus, in P. methanica and Methanomonas methanooxidans the oxygen atom in methanol is derived exclusively from molecular oxygen (Higgins & Quayle, 1970). A feature of the free-radical mechanism for the oxidation of methane by M. capsulatus is that it can readily account for the oxidation of other substrates, e.g. dimethylether, dimethyl carbonate, ethylene, carbon monoxide, bromomethane and ammonia by this organism. Dimethylether is formed under oxygen-limiting conditions (Bryan-Jones & Wilkinson, 1971) possibly because the formation of methylperoxy iron is inhibited allowing the concentration of reactive methyl iron derivatives to rise. When methoxy radicals are formed during the oxidation process, these would then react with the methyl iron to generate dimethyl ether. Oxidation of dimethyl ether could occur by hydrogen abstraction involving iron hydroperoxide to give a free radical. Stabilization of this free radical by co-ordination to iron could be followed by the breakdown of the intermediate, possibly by a concerted, cyclic mechanism. This would liberate formaldehyde and generate a methyl radical stabilized by co-ordination to iron which could then participate in the oxidation pathway.
~ +
t CH30"
CH3OCH3 OOH
0
"CH20CH3+ / / / ~ + H202 CH3 CH20 + ( ~
02
etc
332
D. W. HUTCHINSON,
R.
WHITTENBURY
AND H. DALTON
Dimethyl carbonate which can also be metabolized by M. capsulatus (Bryan Jones & Wilkinson, 1971) could undergo a similar series of reactions following hydrogen-atom abstraction and co-ordination to iron. OOH //~+
CH3OCOOCH3
/~'t-'CH2OC OOCH3 -f H202
elc o
c.2o
.3
Recently it has been shown (Dalton & Whittenbury, 1975) that ethylene is oxidized by M. capsulatus in a reaction which is independent of methane oxidation. The addition of peroxides to ethylene with the formation of epoxides is well known, and in this instance a methylperoxy radical could add to the ethylene to form the peroxide. Solvolysis of the epoxide would lead to ethylene glycol and this could be oxidized further by the organism. CH2= CH2 +CH3OO"
HOCH2CH2OH
-
CH3OOCH2CH2"
H20
CH2--CH2 + CH30"
\o / Unlike saturated hydrocarbons, acetylene forms very stable iron complexes (Nast, 1960). Hence, acetylene might be expected to compete with molecular oxygen for ferrous iron in the initial stages of oxygen activation. This would explain the observed inhibition of methane oxidation by acetylene (Dalton et aL, 1975).
METHANE
OXIDATION
BY
333
M. CAPSULATUS
Carbon monoxide is metabolized in the absence and presence of methane by M. capsulatus, being converted into carbon dioxide. Addition of a methylperoxy radical to carbon monoxide followed by fission of the product could yield carbon dioxide and a methoxy radical, the latter participating in the oxidation chain in the usual manner. CH3OO'+CO'--"CH3OOCO"-"CH30"+C02
Bromo- and chloromethane are metabolized by M. capsulatus while dibromo- and dichloromethane are not (Colby, 1975). This can be rationalized by assuming that the methyl halides react with two equivalents of liganded iron to generate an iron alkyl and an unstable derivation with the halogen linked to iron. In the case of bromomethane, the iron methyl is a component of the oxidation chain proposed above and hence further oxidation could occur. However, dibromomethane would lead to an iron-bromomethyl derivative. While the latter may well give rise to peroxyradical, this would not be able to participate in the oxidation path in the normal manner.
2
-I- CHsBr
+
•
Br
= elc
CH2Br
OOCH2Br
M. capsulatus will oxidize ammonia to nitrite and the first product in this reaction is probably hydroxylamine as is the case with Nitrosomonas (Hofman & Lees, 1953). Hydroxylamine probably arises from a reaction sequence similar to that proposed above for methane oxidation. Hydrogen atom abstraction from ammonia by a peroxy iron derivative followed by reaction of the amino radical with air could lead ultimately to hydroxylamine. A system involving free radical participation in ammonia oxidation by Nitrosomonas has been suggested recently (Hooper & Terry, 1974). The autoxidation of hydroxylamine to nitrite in the presence of cupric ions is known to occur (Hughes & Nicklin, 1970, 1971) accounting for the product observed in this oxidation. T,~.
22
334
D. W . H U T C H I N S O N , R W H I T T E N B U R Y AND H. D A L T O N
OOH //~-I-
HOONH2
NH5
-I-H202
, HI-'NH2
HZO "OONH 2
~,
K
H?02+ HONH z
Cu/O 2
NO 2-
We contend that since the oxidative metabolic reactions of M . capsulatus can be explained by mechanisms which involve free radicals in the initial stages, experimental methods must be devised to check this hypothesis. Experiments with the whole organism are difficult owing to low levels of intermediates and the difficulty of ensuring that reagents added to the medium enter the cell. Cell-free extracts o f M . capsulatus capable of oxidizing methane can now be obtained and e.p.r, studies and radical trapping reactions are being carried out. REFERENCES BRYAN-JoNEs,G. & WmKINSON,J. F. quoted by Wilkinson, J. F. in 21st Symposium for Society for General Microbiology (1971), 15. COLBY,J. (1975). Personal communication. COLBY,J., DALTON,H. & WHrrrENBURY,R. (1975). Biochem. J. 151, 459. DALTON,H. & WHrrTENBtmY,R. (1976). Arch. MicrobioL in press. FBRENCT,T. (1974). F.E.B.S. Lett. 41, 94. GEORGE,P. (1954). J. chem. Soc. 4349. HARWOOD,J. H. & PINT, S. J. (1972). J. appL Bact. 35, 597. HAYAISm,O. (1962). Oxygenases. New York: Academic Press. HAY~aSHI,O. (1969). Ann. Rev. Biochem. 38, 21. I-IIGGINS,I. J. & QUAYL~,J. R. (1970). Biochem. 3. 118, 201. HOV~AN,T. & LEES,H. (1953). Biochem. 3. 54, 279. HoOP~R, A. B. & TERRY,K. R. (1974). J. Bact. 119, 899. HOWARD,J. A. (1973). Free Radicals (J. K. Kochi, ed.) Vol. 2, p. 3. New York: Wiley. HUaLEY, J. H., MrrroN, J. R. & WmKINSON,J. F. (1974). Arch. MikrobioL 95, 365. HUGHES, M. N. & NICKLrN,H. G. (1970). Biochim. biophys. Acta 222, 660. HUQHr-S, M. N. & NXCKLrN~H. G. (1971). J. Chem. Soe. A 164. LEADBE'rTER,E. R. & Fos'r~, J. W. (1959). Nature, Lond. 184, 1428. LrADBETTER,E. R. & FOSTER,J. W. (I960). Arch. MikrobioL, 35, 92. MAZKOVETZ,A. J. (197I). Crit. Revs. MicrobioL 1, 225. NAST, R. (1960). Angew. Chem. 72, 26. PAT~L, R. N. & HOARE,D. S. (1971). J. Bact. 107, 187. Psa'~sor~, J. A. (1971). Arch. Biochem. Biophys. 144, 678.
METHANE OXIDATION BY M. C A P S U L A T U S
335
REED, H. L. (1975). Personal communication. RIBBONS, D. W., HARRISON,J. E. & WADZINSKI,A. M. (1970). Ann. Rev. MicrobioL 24, 135. RIBBONS, D. W. & MICHALOVER,J. L. (1970). FEBS Lett. 11, 41. SOSNOVSKY,G. & BROWN, J. H. (1966). Chem. Rev. 66, 529. ULLRICH, V. (1972). Angew. Chem. (lnternat. Edn.) 11, 701. VAN DIJKEN, J. P. & HARDER, W. (1975). Biotech. Bioeng. 17, 15. WH1TTENBURY,R., PHILLIPS,K. C. & WILKINSON,J. F. (1970)../. gen. Microbiol-61,205. WILKINSON,J. F. (1974). Personal communication.
A Possible Role of Free Radicals in the Oxidation of Methane by Methylococcus capsulatus D. W. HtrrcmNsoN'l"
Department of Molecular Sciences, University of Warwick, Coventry CV4 7AL, England AND
R. WHITrENBURY AND S . DALTON
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, England (Received 30 May 1975) A mechanism involving free radicals is proposed for the oxidation of methane by Methylococcus capsulatus. Removal of a hydrogen atom from methane can be effected by oxygen in the form of a hydroperoxide-iron complex. Subsequent steps in the oxidation of the methyl radical so formed to formaldehyde can be accounted for by known free radical reactions. The oxidation of substrates other than methane (e.g. dimethyl ether, carbon monoxide or ammonia) can be accommodated by variations in the initial free radical mechanism. 1. Introduction
Methane oxidizing bacteria are Gram-negative non-endosporing aerobes which use only C1 compounds, as combined sources of carbon and energy (in the absence of other carbon compounds). It has been tentatively assumed that oxidation of methane is initiated by a mixed-function oxidase (or hydroxylase as it is now known) mechanism similar to that which operates within other n-alkane (C2 +) oxidizing microbes. Indirect evidence for a typical hydroxylase mechanism was provided by (a) the observed incorporation of 1SOz into cell material (Leadbetter & Foster, 1959; Bryan-Jones & Wilkinson, 1971) and into methanol accumulated during methane oxidation (Higgins & Quayle, 1970); and (b). the stimulation by NADH of oxygen consumption by cell-free extracts in th e presence of methane or carbon monoxide (Ribbons & Michalover, 1970; Ferenci, 1974). So far, however, stoichiometric evidence which would support unambiguously a hydroxylase mechanism in cell-free t Addressfor reprint requests. 325
326
D.W.
HUTCHINSON,
R. WHITTENBURY
AND
H. DALTON
extracts of methane oxidisers, identical to that in microbes oxidising C2+ hydrocarbons, is missing. A major problem with cell-free work in this field has been the extreme lability of activity which is limited to 24 hr following preparation. To the experimental evidence above can be added circumstantial evidence to suggest that a conventional hydroxylase function may not be present at all The following observations raise this possibility: (I) Dimethyl ether is excreted and can be utilized as the sole carbon and energy source. Dimethyl carbonate can also be utilized (BryanJones & Wilkinson, 1971). (2) Carbon monoxide and ammonia are oxidized apparently by the same "oxygenase" activity responsible for methane oxidation (Hubley, Mitton & Wilkinson, 1974; Colby, Dalton & Whittenbury, 1975). (3) Other hydrocarbons (e.g. ethane or propane) are oxidized but not used as sole carbon and energy sources. Microbes oxidizing and growing upon n-alkanes (C2+) conversely do not oxidize methane. (4) Methanol, although utilized, is toxic in the medium in concentration as low as 0.01 ~o (v/v). Tolerant strains can be obtained but these are mutants which have very low oxidation rates compared with the wild type (Reed, I975). (5) The molar growth yields obtained with methane are either higher or similar to molar growth yields obtained with methanol. If a mixed function oxidase were involved, oxidation of NADH during the step concerned with the incorporation of oxygen would have to be coupled to ATP synthesis to account for the observed growth yields. To date no activity of this sort has been reported for known hydroxylases. Further consideration of methane oxidation involving sources of NADH for the oxidation step and carbon for growth reveal problems of accounting for growth yields observed, particularly in the Type 1 group of methane oxidisers. Based on the assumption that methane is initially oxidized by a hydroxylase mechanism, Van Dijken & Harder (1975) have concluded that it is necessary to postulate a reverse electron transport system to generate sufficient NADH for cell growth. Under such circumstances, the maximum calculated yield would be 0.91 g cells/g methane. In the absence of an hydroxylase mechanism (and hence a system that requires no input of energy or reductant), the maximum yield possible would be 1.46 g cells/g methane. Since yield values for Methylococcus have been reported to be higher than those calculated for an hydroxylase mechani~sm [e.g. 1.1 (Whittenbury, Phillips & Wilkinson, 1970), 1-0 (Harwood & Pirt, 1972) amongst others of the same order], then the existence of such a mechanism must be in
METHANE OXIDATION
BY M . C A P S U L A T U S
327
some doubt. In addition, since the yield coefficient of cells fixing nitrogen is only 5 7o less than those assimilating nitrate (Dalton & Whittenbury, 1975) and since nitrogen fixation represents a drain on the reducing equivalents of the cells, we find it difficult to rationalize this with the concept of an hydroxylase mechanism in which the cell has to expend energy to produce sufficient NADH for methane assimilation. Furthermore, Van Dijken & Harder have made a number of assumptions, e.g. YArP = 10"5, P/O ratios from NADH = 3 which are acceptable in a theoretical treatment but which need to be justified experimentally. However, one assumption made by these authors which is misleading and which we find difficult to accept is that there is no ATP production resulting from the oxidation of NADH if a reverse electron transport (and hence an hydroxylating) mechanism is functioning. To support this contention Van Dijken & Harder point out that Ribbons, Harrison & Wadzinski (1970) found very low levels of NADH oxidase activity in Methylococcus extracts. In fact, the values for NADH oxidase activity quoted by both Ribbons & Michaelover (1970) and Ferenci (1974) for methane oxidizing extracts were about 40 nmol NADH oxidized/ min/mg protein. Such a value is to be expected for an aerobe that relies extensively on NADH oxidation to obtain its energy for growth. This evidence would suggest that no reverse electron transport operates in methane-oxidizing bacteria and hence that there is no mixed function oxidase. We consider that an alternative to the hydroxylase mechanism for methane oxidation needs to be considered and that a system involving free radicals could possibly account for all the anomalies we have outlined. I-Iydroxylases have been studied for a number of years and enzyme systems which can hydroxylate comparatively unreactive molecules have been detected in both animals and bacteria (Hayaishi, 1962, 1969; Ullrich, 1972). The oxidation of methane possibly via methanol to formaldehyde by methylobacteria has not been studied in such detail, although the first stage in this oxidation can be regarded as similar to hydroxylation by a hydroxylase. A major problem in the oxidation of hydrocarbons and other unreactive molecules by molecular oxygen is the activation of the oxygen so that it will react with C - - H bonds. In some cases it appears that cytochrome P-450 is involved and although the mechanism of activation by this cytochrome is unclear, peroxide and superoxide ions have also been suggested as possible intermediates (Ullrich, 1972). Recent studies on the co-oxidation by Methylococcus capsulatus of substrates in the presence of methane coupled with the circumstantial evidence prompt us to propose a free radical mechanism for the first stages in the oxidation of methane and other substrates by this organism. The proposal of a free radical mechanism is not novel. The appearance
328
D.W.
HUTCHINSON,
R. WHITTENBURY
AND
H. DALTON
CH 4
I
-H"
CHff
o2
C2H4 CH. tO2 . . . .
CH30~
•- ~ ' C H 2 - - C H 2 +
CH30
\ oI / I
I I
CH30 ~ CH5
HOCHEH~H
CH 3" CHsOCH 3 .t i I
I-H* I I
-CH 3"
c.~ocH;
-CH~ 11
-CO2/// /
/
/
c.3~coc. ~ I
"I
CH20
I-H" II CH3OCOOCH3
H20
2e
.coo. I NAD+
~NADH CO2
FIG. I. Possible p a t h w a y s involved in the free radical oxidation o f m e t h a n e . main pathways; ..... , ancillary pathways.
METHANE OXIDATION BY M. CAPSrILATeS
329
of methyl ketones during co-oxidative metabolism of higher alkanes by Pseudomonas methanica prompted the proposal of a free radical mechanism involving alkyl hydroperoxides (Leadbetter & Foster, 1960). Methyl ketones, it was suggested, were formed if the initial oxidative step led to a secondary alcohol and fatty acids were formed if the initial attack produced a primary alcohol. This scheme has been criticized by Markovetz (1971) on the grounds that if the secondary alkyl hydroperoxide is a transitory intermediate then the secondary alcohol need not be formed since the ketone could be formed directly from the hydroperoxide and the alcohol could be the product of reduction of the methyl ketone. However, Markovetz does suggest that a non-specific free radical mechanism could explain the appearance of subterminal oxidation products during the oxidation ofn-decane by P. aeruginosa. Neither scheme mentions the possibility that a free radical mechanism could function in the oxidation of methane. In view of the extreme reactivity of many of the free radical intermediates which might be involved, we propose that some free radicals may be stabilized by co-ordination to a metal and that some reactions may take place between reactants co-ordinated to the same or closely neighbouring metal ions. Methylococcus capsulatus contains both haem and non-haem iron as well as copper, and we propose that molecular oxygen co-ordinated to a metal (e.g. Fe ~I) may be sufficiently activated in the form of an iron(III) hydroperoxide to remove a hydrogen atom from methane. The oxidation of ferrous ions by molecular oxygen has been proposed to take place by hydrogen-atom transfer (George, 1954). This process is catalysed by cupric ions and it may be significant that metal chelating agents which are specific for copper inhibit methane oxidation by M. capsulatus (Wilkinson, 1974). Co-ordination of the ferrous iron with a suitable ligand, e.g. a cytochrome or sulphur-containing protein, may change the energy levels of the iron orbitals and hence assist the hydrogen-atom transfer reaction. For example, cytochrome P-450 is involved in the hydroxylation of camphor by P. putida and electron spin resonance studies indicate that the number of unpaired electrons in the iron atom of cytochrome P-450 increases when camphor is added to the hydroxylating system (Peterson, 1971). Removal of a hydrogen atom from methane by the iron(III) hydroperoxide would generate a methyl radical which could be stabilized by co-ordination to an iron atom. The iron-methyl could then react with molecular oxygen to give a methylperoxy iron derivative, this insertion reaction is known to occur with other metal alkyls (Sosnovsky & Brown, 1966). In addition to the iron-methyl, hydrogen peroxide is formed and this could be broken down homolytically, possibly by a metalloenzyme, to hydroxyl radicals. The latter could also participate in the removal of a
330
D. W . H U T C H I N S O N , R. W H I T T E N B U R Y AND H. D A L T O N
0"~0 02
•
OOH FeE°H2
"
+
WhereYepresents ligonded iron
LFe OHJ
1 CH4
02
+ H2 0 2
hydrogen atom from methane. Dissociation of the methylperoxy iron, followed by dimerization and fission of the dimer to molecular oxygen methoxy radicals (Howard, 1973) would then be the next process to occur. Abstraction of a hydrogen atom from the solvent by the methoxy radical would lead to methanol, alternatively loss of a hydrogen atom from the methoxy radical would generate formaldehyde, which could be oxidized further to formate and finally carbon dioxide by enzymes which do not involve free radicals (Patel & Hoare, 1971).
OOCH3 +
CH 00"
CuII/CuIT c%oo"
CH O0 i
OOCH3
CHsOH-t-'OH
CH20
2 CH30"+02
Although methanol is oxidized by M. capsulatus it is t o x i c at relatively low concentrations (Whittenbury, Phillips & Wilkinson, 1970). It could be
METHANE OXIDATION
331
BY M'. C A P S U L A T U S
argued from the free radical mechanism outlined above that methanol is not on the main path of methane oxidation in this organism but is formed by reduction of formaldehyde, the latter being the essential source of carbon for synthesis. If this were the case, then during the oxidation of methane with 180-labelled oxygen, some of the 180 might be lost from the formaldehyde by exchange with water. While the incorporation of 180 from oxygen into methanol has not been studied in M. capsulatus, in P. methanica and Methanomonas methanooxidans the oxygen atom in methanol is derived exclusively from molecular oxygen (Higgins & Quayle, 1970). A feature of the free-radical mechanism for the oxidation of methane by M. capsulatus is that it can readily account for the oxidation of other substrates, e.g. dimethylether, dimethyl carbonate, ethylene, carbon monoxide, bromomethane and ammonia by this organism. Dimethylether is formed under oxygen-limiting conditions (Bryan-Jones & Wilkinson, 1971) possibly because the formation of methylperoxy iron is inhibited allowing the concentration of reactive methyl iron derivatives to rise. When methoxy radicals are formed during the oxidation process, these would then react with the methyl iron to generate dimethyl ether. Oxidation of dimethyl ether could occur by hydrogen abstraction involving iron hydroperoxide to give a free radical. Stabilization of this free radical by co-ordination to iron could be followed by the breakdown of the intermediate, possibly by a concerted, cyclic mechanism. This would liberate formaldehyde and generate a methyl radical stabilized by co-ordination to iron which could then participate in the oxidation pathway.
~ +
t CH30"
CH3OCH3 OOH
0
"CH20CH3+ / / / ~ + H202 CH3 CH20 + ( ~
02
etc
332
D. W. HUTCHINSON,
R.
WHITTENBURY
AND H. DALTON
Dimethyl carbonate which can also be metabolized by M. capsulatus (Bryan Jones & Wilkinson, 1971) could undergo a similar series of reactions following hydrogen-atom abstraction and co-ordination to iron. OOH //~+
CH3OCOOCH3
/~'t-'CH2OC OOCH3 -f H202
elc o
c.2o
.3
Recently it has been shown (Dalton & Whittenbury, 1975) that ethylene is oxidized by M. capsulatus in a reaction which is independent of methane oxidation. The addition of peroxides to ethylene with the formation of epoxides is well known, and in this instance a methylperoxy radical could add to the ethylene to form the peroxide. Solvolysis of the epoxide would lead to ethylene glycol and this could be oxidized further by the organism. CH2= CH2 +CH3OO"
HOCH2CH2OH
-
CH3OOCH2CH2"
H20
CH2--CH2 + CH30"
\o / Unlike saturated hydrocarbons, acetylene forms very stable iron complexes (Nast, 1960). Hence, acetylene might be expected to compete with molecular oxygen for ferrous iron in the initial stages of oxygen activation. This would explain the observed inhibition of methane oxidation by acetylene (Dalton et aL, 1975).
METHANE
OXIDATION
BY
333
M. CAPSULATUS
Carbon monoxide is metabolized in the absence and presence of methane by M. capsulatus, being converted into carbon dioxide. Addition of a methylperoxy radical to carbon monoxide followed by fission of the product could yield carbon dioxide and a methoxy radical, the latter participating in the oxidation chain in the usual manner. CH3OO'+CO'--"CH3OOCO"-"CH30"+C02
Bromo- and chloromethane are metabolized by M. capsulatus while dibromo- and dichloromethane are not (Colby, 1975). This can be rationalized by assuming that the methyl halides react with two equivalents of liganded iron to generate an iron alkyl and an unstable derivation with the halogen linked to iron. In the case of bromomethane, the iron methyl is a component of the oxidation chain proposed above and hence further oxidation could occur. However, dibromomethane would lead to an iron-bromomethyl derivative. While the latter may well give rise to peroxyradical, this would not be able to participate in the oxidation path in the normal manner.
2
-I- CHsBr
+
•
Br
= elc
CH2Br
OOCH2Br
M. capsulatus will oxidize ammonia to nitrite and the first product in this reaction is probably hydroxylamine as is the case with Nitrosomonas (Hofman & Lees, 1953). Hydroxylamine probably arises from a reaction sequence similar to that proposed above for methane oxidation. Hydrogen atom abstraction from ammonia by a peroxy iron derivative followed by reaction of the amino radical with air could lead ultimately to hydroxylamine. A system involving free radical participation in ammonia oxidation by Nitrosomonas has been suggested recently (Hooper & Terry, 1974). The autoxidation of hydroxylamine to nitrite in the presence of cupric ions is known to occur (Hughes & Nicklin, 1970, 1971) accounting for the product observed in this oxidation. T,~.
22
334
D. W . H U T C H I N S O N , R W H I T T E N B U R Y AND H. D A L T O N
OOH //~-I-
HOONH2
NH5
-I-H202
, HI-'NH2
HZO "OONH 2
~,
K
H?02+ HONH z
Cu/O 2
NO 2-
We contend that since the oxidative metabolic reactions of M . capsulatus can be explained by mechanisms which involve free radicals in the initial stages, experimental methods must be devised to check this hypothesis. Experiments with the whole organism are difficult owing to low levels of intermediates and the difficulty of ensuring that reagents added to the medium enter the cell. Cell-free extracts o f M . capsulatus capable of oxidizing methane can now be obtained and e.p.r, studies and radical trapping reactions are being carried out. REFERENCES BRYAN-JoNEs,G. & WmKINSON,J. F. quoted by Wilkinson, J. F. in 21st Symposium for Society for General Microbiology (1971), 15. COLBY,J. (1975). Personal communication. COLBY,J., DALTON,H. & WHrrrENBURY,R. (1975). Biochem. J. 151, 459. DALTON,H. & WHrrTENBtmY,R. (1976). Arch. MicrobioL in press. FBRENCT,T. (1974). F.E.B.S. Lett. 41, 94. GEORGE,P. (1954). J. chem. Soc. 4349. HARWOOD,J. H. & PINT, S. J. (1972). J. appL Bact. 35, 597. HAYAISm,O. (1962). Oxygenases. New York: Academic Press. HAY~aSHI,O. (1969). Ann. Rev. Biochem. 38, 21. I-IIGGINS,I. J. & QUAYL~,J. R. (1970). Biochem. 3. 118, 201. HOV~AN,T. & LEES,H. (1953). Biochem. 3. 54, 279. HoOP~R, A. B. & TERRY,K. R. (1974). J. Bact. 119, 899. HOWARD,J. A. (1973). Free Radicals (J. K. Kochi, ed.) Vol. 2, p. 3. New York: Wiley. HUaLEY, J. H., MrrroN, J. R. & WmKINSON,J. F. (1974). Arch. MikrobioL 95, 365. HUGHES, M. N. & NICKLrN,H. G. (1970). Biochim. biophys. Acta 222, 660. HUQHr-S, M. N. & NXCKLrN~H. G. (1971). J. Chem. Soe. A 164. LEADBE'rTER,E. R. & Fos'r~, J. W. (1959). Nature, Lond. 184, 1428. LrADBETTER,E. R. & FOSTER,J. W. (I960). Arch. MikrobioL, 35, 92. MAZKOVETZ,A. J. (197I). Crit. Revs. MicrobioL 1, 225. NAST, R. (1960). Angew. Chem. 72, 26. PAT~L, R. N. & HOARE,D. S. (1971). J. Bact. 107, 187. Psa'~sor~, J. A. (1971). Arch. Biochem. Biophys. 144, 678.
METHANE OXIDATION BY M. C A P S U L A T U S
335
REED, H. L. (1975). Personal communication. RIBBONS, D. W., HARRISON,J. E. & WADZINSKI,A. M. (1970). Ann. Rev. MicrobioL 24, 135. RIBBONS, D. W. & MICHALOVER,J. L. (1970). FEBS Lett. 11, 41. SOSNOVSKY,G. & BROWN, J. H. (1966). Chem. Rev. 66, 529. ULLRICH, V. (1972). Angew. Chem. (lnternat. Edn.) 11, 701. VAN DIJKEN, J. P. & HARDER, W. (1975). Biotech. Bioeng. 17, 15. WH1TTENBURY,R., PHILLIPS,K. C. & WILKINSON,J. F. (1970)../. gen. Microbiol-61,205. WILKINSON,J. F. (1974). Personal communication.
