Biological methylation of mercury can occur by many routes, says biochemist W o o d Though it is an acute poison, inorganic mercury is relatively innocuous when compared with alkyl mercury compounds. Alkyl mercurials account for only 4 % of annual mercury use in the U.S., and are largely confined to use as seed dressings. It was, therefore, the discovery that inorganic mercury could be alkylated to methylmercury in natural systems that pointed up the present dimensions of the mercury pollution problem. Broken thermometers, plastics tainted with mercury catalysts, aqueous effluent from chlor-alkali plants, paints (in which mercury protects against mildew), papermaking (where phenylmercury compounds are used as slimicides), and the com bustion of coal and oil all release mercury to the environment —and all of this mercury can be methylated. Dr. John M. Wood, a biochemist at the University of Illinois, Urbana, was one of the first to demonstrate the mechanism for methylation of mercury in natural systems, and he has now worked out many of the reactions involved. In 1964, Jack Halpern and J. P. Maher at the University of Chicago showed that methylpentacyanocobaltate—a model compound for vitamin B12—would react wtih mercuric ion to give methylmercury. Thus, Dr. Wood reasoned, Bi 2 -containing enzyme systems should be capable of this synthesis in biological systems. Of the three major coenzymes known to be available for methyl transfer reactions in biological sys tems—S-adenosylmethionine (SAM), N5-methyltetrahydrofolate derivatives, and methylcorrinoid derivatives—the first two involve methyl group transfer as a carbonium ion. There fore, Dr. Wood concludes, only the methylcorrinoids are cap able of methyl transfer, since they are theoretically capable of transferring methyl groups as carbanions (CH3"), carbonium ions (CH3+), or radicals (CH3·): Scheme I
H
V
H
4 ν+
Hé Λ \
\
\
St'* β
CHf CH^
wf
CL vuAueio. oj- baAZL· c a n coMotUtûfo tô
In 1968, Dr. Wood and coworkers showed that methylcobalamin was capable of synthesizing both monomethylmercury and dimethylmercury in enzymic or nonenzymic systems, with the relative proportion of the products dependent on the concentration of mercuric ion. This research proved that the biosynthesis of methylmercury could occur under anaerobic conditions. Further kinetic experiments have now shown that Hg2+ reacts with methylcorrinoids in two ways. The methyl transfer is pH-dependent, and does not proceed to stoichiometry unless the ratio of Hg2+ to methylcorrinoid is 2 : 1 . This stoichiometry, Dr. Wood says, suggests that one mercuric ion must react with one methylcorrinoid molecule (rapidly) before CH3~ is subjected to electrophilic attack by a second mercuric ion (slowly). This nonenzymic reaction does not occur in the presence of Hg22+ or Hg°. Therefore, Dr. Wood concludes,
24
C&EN JULY 5, 1971
CH3
CH /
I
N
C
©
t . Β
^K Η Η
-Hf
η γ/
Η,
H20
-Η
Β '
is still attempting to determine why two mercuric ions are necessary for displacement of one CH3" in this synthesis. Methylcorrinoids are also implicated in mercury methylation by three enzyme systems, Dr. Wood says: • Cobalamin-dependent N5-methyltetrahydrofolate-homocysteine transmethylase (methionine synthetase). • Acetate synthetase. • Methane synthetase. Methionine synthetase. A number of microorganisms, in cluding a mutant of the fecal organism Escherichia coli, use the cobalamin-dependent methionine synthetase to synthesize the essential amino acid methionine. This enzyme is present in some aerobes and anaerobes, as well as in mammalian liver. In this system methylcobalamin binds to the methionine synthetase apoenzyme to give the active enzyme-substrate complex: Scheme III
6= -fcouie {UUQUOSL· 5, fe-oL/v*çfliuG&M2(>n^
ha
Scheme II
CH,
5H
+ \ Co / V u \(
\ i 3> \ r - 2 t Lo x —»Co x -t 6
this reaction would be predominant in those aerobic organisms that use methylcorrinoids in their intermediary metabolism, since under anaerobic conditions 2Hg2+ plus two electrons yields Hg22+, and Hg22+ plus two electrons yields 2Hg°. Nuclear magnetic resonance and ultraviolet-visible spectroscopic data suggest that no valence change occurs for the cobalt atom (Co3+) during methyl transfer to Hg2+, proving that the methyl group is transferred as CH3~ (scheme II). Dr. Wood
+
S-CH,
5AM
(CHA ^ ) Ç ô t
+
(CH2)2
^ N H ? - C H - C 0 0 e ^ N H ? - C H -COO* Nff-CH,-THF
THF
For this reaction, catalytic amounts of SAM are required, according to Dr. Wood, and N5-methyltetrahydrofolate regenerates the methionine synthetase enzyme-substrate complex. Aerobic microorganisms and facultative anaerobes which use the cobalamin-dependent methionine synthetase enzyme are capable of synthesizing methylmercury, and mammalian liver will catalyze the reaction slowly, Dr. Wood notes. Scheme IV
Η Η
fa)
\/ Wfi 0 H3»^ » Co + CH5Hg©
CH,
(W μ
ν
Η
FADH2-
γ·9
tFAP
CH 5
(c)
X-CHj-jfjV^X-H+OV
^• x Co + f+THF
^^MkHjTHF-
/f \
fa)CH3Hf + 5H
(ÇHA
CH 3 -H 9
NHf-CH-COtf»
5 + H® (CH2)2 NHÎ-CHC0CP
quired in catalytic amounts. Dr. Wood does not know why. The use of isotopes and photolysis control experiments prove that methyl transfer in methane synthetase occurs by a radical mechanism, Dr. Wood says. When methylcorrinoids are photolyzed under anaerobic conditions, homolysis of the Co-C bond occurs to give Co2+ and methyl radicals: Scheme VII
Η Η CH 3
RADH2= neducciL^avmt-aolflUÛii a^cie^iufe Electrophilic attack on the methylcobalamin-methionine synthetase complex yields methylmercury (scheme IV). Reduction of the resulting Co3+ gives Co+, which is remethylated by CH3+ transfer from N5-methyltetrahydrofolate. The CH3Hg+ that is formed removes the substrate homocysteine from the active site of the enzyme. Some methyl transfer may occur, he adds, to give methylthiomethylmercury (CH3HgSCH3) also. Acetate synthetase. Clostridium thermoaceticium and Clostridium sticklandii, anaerobic organisms that synthesize acetic acid from carbon dioxide, are other sources of methylmercury, Dr. Wood notes: Scheme V
//f
Η,Ο
\
0
6
/t \ &
Since experimental data provide a basis for CH3· transfer, and since the redox potential required for growth of these strict anaerobes is less than —400 mv., then any inorganic mercury salt added to the methane synthetase enzyme system would be reduced to Hg°, Dr. Wood finds. Dimethylmercury is then synthesized by CH3' addition to Hg°: Scheme VIII
CH } 2)Co 3 > / X
•2)C CÔZ^(CH3)2H3
+
\
6
OC^+CH3COOH
It seems feasible, Dr. Wood says, that Hg2+ may be transported across cell membranes by these anaerobes, reduced to metallic mercury, and then methylated as in scheme VIII. Dimethylmercury would readily diffuse from the microbial cells, he adds, and if the pH of the sediments was alkaline, this compound would be released to the water. If the pH was acid, would be converted to monomethylmercury and methane: Scheme IX
NADP+ T H F ^ Y - ^ C ô r ^ VNADPH NAD Ρ = ριυίίψί
WÙZX&MJOJMÙJÎSL - OAÛJÙMS,
NADPH = n
