Organometals and Organometalloids - American Chemical Society

7 Anaerobic and Aerobic Alkylation of Arsenic BARRY C. McBRIDE, HEATHER MERILEES, WILLIAM R. CULLEN, and WENDY PICKETT

Downloaded by UNIV LAVAL on September 23, 2015 | http://pubs.acs.org Publication Date: January 12, 1979 | doi: 10.1021/bk-1978-0082.ch007

Departments of Microbiology and Chemistry, University of British Columbia, Vancouver, British Columbia, V6T 1W5 Canada

Arsenic and i t s derivatives are important as herbicides and are used in, or are by-products of, many industrial processes. Large quantities of this potentially toxic compound are mobilized and placed i n new environments where they accumulate in concentrations which may exceed the normal arsenic burden of these ecosystems. The microflora i n these environments possess the metabolic machinery to transform these compounds into gaseous arsines. Challenger (1) described the formation of trimethylarsine by fungi. Cox and Alexander (2) have shown that s o i l organisms w i l l produce trimethylarsine and McBride and Wolfe (3) reported that the anaerobic methane bacteria synthesized dimethylarsine. A consequence of this microbial activity i s the modification of arsenic to new compounds possessing different chemical, physical, and biological properties. Methanogenic B a c t e r i a The methanogenic b a c t e r i a a r e a unique group of m i c r o organisms which produce methane as their principal m e t a b o l i c end product. They a r e found in l a r g e numbers in anaerobic ecosystems when o r g a n i c matter is decomposing. As a group they are morphol o g i c a l l y d i v e r s e , embracing c o c c a l , b a c i l l a r y and s p i r a l forms. They a r e extremely s e n s i t i v e t o 0 » a f a c t o r which has c o n t r i b uted t o our l i m i t e d understanding o f t h e i r biochemical a c t i v i t i e s . A r e s t r i c t e d number o f s u b s t r a t e s can be reduced t o methane these i n c l u d e : C 0 , formate, a c e t a t e , and methanol. Hydrogen i s the p r e f e r r e d source o f e l e c t r o n s . The p e r t i n e n t c h a r a c t e r i s t i c s of the methane b a c t e r i a a r e summarized i n Table I . There a r e s e v e r a l (4-8) reviews o f these organisms which d e a l w i t h t h e i r i s o l a t i o n , c h a r a c t e r i z a t i o n and b i o c h e m i s t r y . The methane b a c t e r i a f u n c t i o n as the t e r m i n a l members o f the 2

2

0-8412-0461-6/78/47-082-094$05.50/0 © 1978 American Chemical Society In Organometals and Organometalloids; Brinckman, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

7.

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Table I . C h a r a c t e r i s t i c s of the Methanogenic B a c t e r i a Organisms

Habitat

Morphology

Substrates

Methanobacterium ruminantium

rumen sludge

coccus t o short rods

H2+CO2

Methanobacterium s t r a i n M.oH

mud sludge

irregularly curved rods

H2+CO2

Methanobacterium formicicum

mud, sludge

irregularly curved rods

H2+CO2

Methanobacterium mobilie

rumen

short r o d , motile

H2"KX)2

Methanosarcina barkerii

mud, sludge

Methanococcus vannielii

mud

Methanospirilium hungatii

mud,sludge

sarcina

formate

formate H2+CO2

methanol, acetate m o t i l e coccus

H2+CO2

formate

mud, sludge Methanobacterium thermoautotrophicum hot s p r i n g s

spirillum

H2+CO2

formate irregularly curved r o d

H2+CO2

formate

In Organometals and Organometalloids; Brinckman, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.


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anaerobic food c h a i n , scrubbing the environment of p o t e n t i a l l y t o x i c components and r e l e a s i n g a non-toxic end product which e v e n t u a l l y escapes i n t o oxygenated environments. A g e n e r a l i z e d scheme (6) i l l u s t r a t i n g the e s s e n t i a l steps r e q u i r e d to synthesize a molecule of methane i s summarized i n F i g . 1. The scheme accounts f o r a l l the known methane precursors and i n d i c a t e s the r e a c t i o n s r e q u i r e d to form a completely reduced carbon molecule. Compound X i s a c a r r i e r which may represent one or more molecular s p e c i e s . Our understanding of the steps i n methane b i o s y n t h e s i s i s sketchy and i s almost e n t i r e l y l i m i t e d to the t e r m i n a l methyl t r a n s f e r r e a c t i o n s . No intermediates between CO2 and X - C H 3 , have been i d e n t i f i e d . The c a r r i e r molecule X has been p o s t u l a t e d to be r e q u i r e d because no f r e e reduced C - l intermediates have been i d e n t i f i e d . Progress has been made i n understanding the mechanism of the methyl t r a n s f e r r e a c t i o n s . B l a y l o c k and Stadtman (9) demonstrated that methyl cobalamin would serve as a s u b s t r a t e f o r methane b i o s y n t h e s i s i n c e l l e x t r a c t s of Methanosarcina b a r k e r i i . Subsequently Wo l i n et_ a l (10) found that methylcobalamin was a s u b s t r a t e f o r methane b i o s y n t h e s i s i n Methanobacterium o m e l i a n s k i i . B l a y l o c k was able to r e s o l v e the Methanosarcina (11) system i n t o a number of components,one of which was a c o r r i n o i d c o n t a i n i n g p r o t e i n . Wood (12) was s u c c e s s f u l i n i s o l a t i n g a cobalamin c o n t a i n i n g p r o t e i n from M. o m e l i a n s k i i . The p r o t e i n s t i m u l a t e d CHi+ production when added to c e l l e x t r a c t s (13) of the organism. These s t u d i e s suggested that B12 might be the X f a c t o r i n v o l v e d i n methyl t r a n s f e r . This seemed p a r t i c u l a r l y f e a s i b l e i n l i g h t of the r o l e of C H 3 - B 1 2 i n the CH3 t r a n s f e r r e a c t i o n s l e a d i n g to methionine b i o s y n t h e s i s . U n f o r t u n a t e l y , Wood s c u l t u r e was found to c o n s i s t of two organisms; a. methanogen (Methanobacterium s t r a i n M.oH) which reduced CO2 to CHi* and a second organism which s u p p l i e d e l e c t r o n s f o r methanogenesis by o x i d i z i n g ethanol to acetate and hydrogen (14). R e s u l t s from i n v e s t i g a t i o n s w i t h the mixed c u l t u r e must be viewed w i t h c a u t i o n because i t i s impossible to i d e n t i f y w i t h c e r t a i n t y the g e n e t i c o r i g i n of the B12 p r o t e i n which s t i m u l a t e d methanogenesis. Biochemical s t u d i e s have been complicated by the l a b i l i t y of the e x t r a c t s and u n t i l a few years ago i t was not p o s s i b l e to f r a c t i o n a t e the c e l l s or r e s o l v e them f o r s p e c i f i c components of the methane s y n t h e s i z i n g system. As a consequence i t was necessary to use s t i m u l a t i o n of CHif b i o s y n t h e s i s as the assay f o r c o n s t i t u e n t s which had been f r a c t i o n a t e d from other e x t r a c t s . In t h i s type of assay the r e a c t i o n mixture was complete and would support a l i m i t e d amount of CHi* b i o s y n t h e s i s . Fractionated c e l l e x t r a c t was then added to the r e a c t i o n mixture and i f i t s t i m u l a t e d CHi* production i t was i m p l i c a t e d i n the t e r m i n a l methyl t r a n s f e r r e a c t i o n s . A d i f f i c u l t y w i t h t h i s type of assay i n a complex m u l t i s t e p r e a c t i o n i s that one can only measure s t i m u l a t i o n of a r a t e l i m i t i n g r e a c t i o n . In methane b i o s y n t h e s i s , 1


7.

97

Alkyhtion of Arsenic

MCBRIDE E T A L .

s t i m u l a t i o n could r e s u l t from i n c r e a s e d e l e c t r o n t r a n s p o r t , a c t i v a t i o n o f ATP,or t r a n s f e r of methyl groups. Thus the i m p l i c a t i o n o f a c o r r i n o i d p r o t e i n i n CHi» b i o s y n t h e s i s by Μ· o m e l i a n s k i i was based on r a t h e r tenuous evidence, but c e r t a i n l y the best evidence that could be developed a t that time. The isolâtion,of the pure c u l t u r e o f M.oH f a c i l i t a t e d the study of methanogenesis; c e l l e x t r a c t s were not as l a b i l e and c a t a l y z e d CHi* formation a t an i n c r e a s e d r a t e . I n a d d i t i o n these


e x t r a c t s reduced C 0

t o CHi*

2

(7) .

Attempts t o i s o l a t e the c o r r i n o i d p r o t e i n from M.oH have been unsuccessful (15). I n f a c t no B 1 2 c o n t a i n i n g enzymes appear to be present i n t h i s organism. The red p r o t e i n i s o l a t e d by Wood was shown t o be synthesized by the non-methanogenic contaminant (15) and thus could not be a p a r t o f the i n v i v o methane s y n t h e s i z i n g system. The i n a b i l i t y t o f i n d a c o r r i n o i d p r o t e i n cast some doubt on the importance o f C H 3 - B 1 2 i n t e r m i n a l methyl transfer reactions. The question was answered w i t h the discovery o f the methyl donor, C H 3 - C 0 M . CoM i s a low m o l e c u l a r weight, heat s t a b l e c o f a c t o r found i n a l l methane b a c t e r i a that have been examined (16). Chemically CoM i s 2,2 - d i t h i o d i e t h a n e s u l f o n i c a c i d (17). I t can be reduced and methylated c h e m i c a l l y o r b i o l o g i c a l l y t o form 2-(methylthio) e t h a n e s u l f o n i c a c i d . I n the presence of c e l l e x t r a c t , ATP, and H ,CH -CoM i s r e d u c t i v e l y cleaved t o y i e l d CH^ and CoM ( F i g . 2 ) . E x t r a c t s can be r e s o l v e d f o r CoM by anaerobic d i a l y s i s and w i l l synthesize CHi* only i f they a r e s u p p l i e d w i t h CoM. I t seems reasonable t o assume that Βχ2 i s n o t i n v o l v e d i n CHi» b i o s y n t h e s i s i n MoH, and that t h i s r e a c t i o n i s c a t a l y z e d by CoM. I t i s not c l e a r a t t h i s time whether Methanosarcina depends on B 1 2 , as t h i s organism has been shown t o possess CoM i n a d d i t i o n t o a c o r r i n o i d p r o t e i n . P o s s i b l y t h i s organism possesses 2 methane s y n t h e s i z i n g pathways,one dependent and one independent of Β 1 2 · f

2

3

A l k y l a t i o n o f Metals M.oH has been i m p l i c a t e d as an organism which w i l l a l k y l a t e mercury (18) and a r s e n i c ( 3 ) . A l k y l a t i o n o f metals by M.oH could t h e o r e t i c a l l y occur a t any b i o s y n t h e t i c step which generated a C - l group. Because the methanogenic b a c t e r i a process l a r g e numbers of C - l u n i t s i t i s not unreasonable to assume that the CH^ b i o s y n t h e t i c pathway could be important i n these r e a c t i o n s , and that CoM would be a molecule of i n t e r e s t . I t would be unwise t o f o r g e t that methyl groups must be generated f o r the s y n t h e s i s of methionine and that intermediates i n t h i s r e a c t i o n could be i n v o l v e d i n metal a l k y l a t i o n . Two methionine b i o s y n t h e t i c pathways operate i n b a c t e r i a . One system i n v o l v e s t r a n s f e r o f a methyl group from N -methyl-tetrahydrofolic acid (N , CH3THFA) to B and then t o homocysteine. The methylation of homocysteine r e q u i r e s c a t a l y t i c 5

5

1 2


ORGANOMETALS

98

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amounts of S-adenosyl methionine (SAM). The second system r e q u i r e s N , CH -THFA t r i - g l u t a m a t e and SAM. The i n a b i l i t y to f i n d a c o r r i n o i d p r o t e i n i n M.oH suggests that the l a t t e r mechanism may operate i n these organisms. 5

3


Arsine Biosynthesis C e l l e x t r a c t s of M.oH produce a s t r o n g g a r l i c odor when they are incubated w i t h arsenate. The f o l l o w i n g s e c t i o n of t h i s paper w i l l d e a l w i t h the b i o c h e m i c a l s t u d i e s which l e d to the i d e n t i f i c a t i o n of the g a r l i c s m e l l i n g compound and to a scheme f o r i t s b i o s y n t h e s i s . This w i l l be f o l l o w e d by a study of a r s e n i c t r a n s f o r m a t i o n i n n a t u r a l anaerobic systems . A l k y l a r s i n e Assays. The r e a c t i o n v e s s e l and c o n d i t i o n s used f o r the b i o s y n t h e s i s of a l k y l a r s i n e s were s i m i l a r to those described f o r methane b i o s y n t h e s i s (19). To t r a p a l k y l a r s i n e s the r e a c t i o n v e s s e l was connected by polyethylene tubing to a g l a s s tube which contained 2 mL of 2M HNO3. The t r a p p i n g tube was placed i n an e t h a n o l - i c e bath. A slow stream of H2 was passed i n t o the r e a c t i o n f l a s k and the v o l a t i l e a l k y l a r s i n e s were swept i n t o the n i t r i c a c i d , where they were condensed and trapped by o x i d a t i o n to n o n - v o l a t i l e a c i d s (20). The contents of the t r a p were assayed f o r a r s e n i c by atomic a b s o r p t i o n spectrometry and f o r C by counting i n Bray's s c i n t i l l a t i o n f l u i d . The i s o t o p e , *As was used to f o l l o w the formation of v o l a t i l e a l k y l a r s i n e . E x t r a c t s were incubated w i t h [ A s ] N a 2 H A s 0 i * and the v o l a t i l e methylated As was measured. Advantage was taken of the property of a l k y l a r s i n e s to r e a c t w i t h the red-rubber serum stoppers which were used to s e a l the r e a c t i o n f l a s k s . At the a p p r o p r i a t e times the r e a c t i o n mixture was i n a c t i v a t e d by h e a t i n g on a steam cone. The f l a s k s were then incubated f o r an a d d i t i o n a l 20 min to ensure t h a t a l l the v o l a t i l e d i m e t h y l a r s i n e was adsorbed onto the rubber stopper. The serum stopper was removed from the f l a s k , r i n s e d i n water, cut i n h a l f , and placed i n a s c i n t i l l a t i o n v i a l together w i t h 15 mL of Bray's s c i n t i l l a t i o n f l u i d . T h i s mixture was e i t h e r counted d i r e c t l y , as the rubber stopper d i d not quench the high-energy 3 p a r t i c l e s emitted by ks, or the stopper was removed a f t e r the **As had been leached from the rubber (1 h r . ) . The rubber stopper technique was found to be an e f f i c i e n t and s p e c i f i c means of s e p a r a t i n g [ C] dime thy l a r s i n e from [ C]CHi . lh

7l

7lf

7lf

7k

llf

1If

t

Requirements f o r B i o s y n t h e s i s . Conditions f o r the b i o s y n t h e s i s of a l k y l a r s i n e d e r i v a t i v e s are d e s c r i b e d i n Table I I . In these experiments c e l l e x t r a c t s were incubated under anaerobic c o n d i t i o n s w i t h [ * A s ] N a 2 H A s 0 i , and the formation of v o l a t i l e *ks compounds was measured. A methyl donor, H 2 , ATP, and arsenate were r e q u i r e d f o r the r e a c t i o n ; w i t h the exception of arsenate these same components are r e q u i r e d i n the CHi*s y n t h e s i z i n g system. A t o t a l of 34 yg of a r s e n i c was found i n 7l

f

7t


7.

MCBRiDE E T A L .

Alkyfotion of Arsenic

99

an a l k y l a r s i n e t r a p l i n k e d to a r e a c t i o n f l a s k which contained arsenate and c e l l e x t r a c t , p r o v i d i n g evidence that a v o l a t i l e a l k y l a r s i n e was b e i n g s y n t h e s i z e d . V o l a t i l e a l k y l a r s i n e compounds were not d e t e c t e d i n a s i m i l a r c o n t r o l r e a c t i o n mixture which contained arsenate and b o i l e d e x t r a c t .


S u b s t r a t e s . C H 3 - B 1 2 and CO2 were shown to be methyl donors f o r a l k y l a r s i n e s y n t h e s i s . Arsenate, a r s e n i t e and m e t h y l a r s o n i c a c i d were reduced to d i m e t h y l a r s i n e i n the presence of a C - l donor. C a c o d y l i c a c i d was reduced i n the absence of a C - l donor. M e t h y l a r s o n i c a c i d was found i n e x t r a c t s incubated w i t h AsOi* "and a C - l donor. Whole C e l l s . The a b i l i t y of whole c e l l s to s y n t h e s i z e a l k y l a r s i n e i s shown i n F i g u r e 3. I n t h i s experiment a sample of an a c t i v e l y growing c u l t u r e was removed a n a e r o b i c a l l y from a 12-L fermentor and was incubated a n a e r o b i c a l l y under a H : C 0 atmosphere. 2

2

A n a l y s i s of the A l k y l a t e d A r s i n e . The s t r u c t u r e of the a l k y l a r s i n e formed i n c e l l e x t r a c t s was i n d i c a t e d by two experimental procedures. I n one procedure N a 2 H A s 0 i f was incubated w i t h [ C]methylcobalamin, and the trapped a l k y l a r s i n e was analyzed f o r a r s e n i c and C. The r e s u l t s of two such experiments are presented i n Table I I I . D i s s o l v e d [^CjCHt, was removed by b u b b l i n g CHi* through the t r a p p i n g s o l u t i o n a f t e r i t had been disconnected from the r e a c t i o n f l a s k . The number of methyl groups i n the trap was c a l c u l a t e d from the s p e c i f i c a c t i v i t y of [ C ] C H 3 - B i 2 . T h i s v a l u e was d i v i d e d by the amount of a r s e n i c to o b t a i n a methyl group to a r s e n i c r a t i o . The r a t i o s obtained (1.8:1 and 1.9:1) i n d i c a t e that the compound i s dimethylarsine. These r e s u l t s were s u b s t a n t i a t e d by a d o u b l e - l a b e l i n g experiment i n which [ *As]Na2HAs0if and [ C]methylcobalamin were s u b s t r a t e s . The r e s u l t s of two such experiments are shown i n Table IV. A l k y l a r s i n e was separated from contaminating [ C]CHi by t r a p p i n g i n a rubber serum stopper as d e s c r i b e d p r e v i o u s l y . The s p e c i f i c a c t i v i t y of the s u b s t r a t e s was used to c a l c u l a t e the micromoles of a r s e n i c as w e l l as methyl groups. The r a t i o s of methyl groups to a r s e n i c (2.1:1 and 1.9:1) suggest that the g a r l i c - s m e l l i n g compound i s d i m e t h y l a r s i n e . A pathway f o r d i m e t h y l a r s i n e s y n t h e s i s has been proposed (3) F i g . 4. Arsenate i s f i r s t reduced to a r s e n i t e which i s then methylated to form m e t h y l a r s o n i c a c i d . T h i s compound was found i n r e a c t i o n mixtures when e x t r a c t s were incubated w i t h e i t h e r arsenate o r a r s e n i t e and C H 3 - B 1 2 . M e t h y l a r s o n i c a c i d i s reduced and methylated to form d i m e t h y l a r s i n i c a c i d . The a c i d i s then reduced to form dime t h y l a r s i n e . A l l i n t e r m e d i a t e s have been shown to be converted to d i m e t h y l a r s i n e by c e l l e x t r a c t s . T h i s pathway i s based on s t u d i e s i n which C H 3 - B 1 2 was used as methyl llf

llf

7l

llf

llf

f


ORGANOMETALS

A N D ORGANOMETALLOIDS

CO + XH a

ι X-COOH

ι ». X-CHO

1 I>

CH,OH

XCH OH 2

h


ChLCOOH

X-CH

Figure 1. Possible pathway for methane biosynthesis. C-2 of ace tate is preferentially reduced to methane.

3

H

2

X + CH (-S-CH CH S03 ) 2

1

2

h

CH As 0 3

3

-o "-


2

9p f_>

3

·· τ τ τ CH As O

2~

CH

i l J

3

2

V Ϊ (CH ) As 0 3

3

+ 3

>

2e ——f

y _ (CH ) As 0 V

3

2

2

2e ••III _ > (CH ) As 0 3

2

. ·· I I I (CH )As 3

-o 2

T h i s scheme was proposed on the b a s i s of the a b i l i t y of S. b r e v i c a u l i s t o produce t r i m e t h y l a r s i n e from a l l the arsenic(V) p r e cursors and t o produce l a b e l e d t r i m e t h y l a r s i n e from a r s e n i t e when C l a b e l e d D,L-methionine, *CH SCH CH CH(NH )C00H, was added to the c u l t u r e medium. The mechanism i s e s s e n t i a l l y the same as that of F i g . 4 except that the methyl donor i s i d e n t i f i e d as the chemically reasonably (29) methyl carbonium i o n and that the r e a c t i o n pro ceeds f u r t h e r , before the f i n a l r e d u c t i o n , i n the aerobic system. Challenger r e p o r t s that crude c e l l e x t r a c t s of S. b r e v i c a u l i s do not produce t r i m e t h y l a r s i n e when exposed t o a r s e n i c a l s . T h i s and other experiments (1) i n d i c a t e d that b i o l o g i c a l methylation by t h i s organism i s confined t o the mould c e l l , and does not take p l a c e i n the medium. I n t h i s connection some recent work on c e l l e x t r a c t s o f C. humicola a r e o f considerable i n t e r e s t (30). I t i s found that when the e x t r a c t i s incubated w i t h As l a b e l e d arsenate, SAM as methyl source, and NADPH as reducing agent, and the r e s u l t i n g supernatant l i q u i d a p p l i e d t o a Dowex 1 i o n exchange column, s e v e r a l d i s t i n c t a r s e n i c c o n t a i n i n g f r a c t i o n s can be e l u t e d as shown i n F i g . 7. These can be i d e n t i f i e d f o l l o w i n g e l e c t r o p h o r e s i s and autoradiography as being l a r g e l y a r s e n i t e , c a c o d y l i c a c i d , methylarsonic a c i d , and arsenate i n f r a c t i o n s 1 t o 4 r e s p e c t i v e l y . Experiments of t h i s s o r t can be r e f i n e d f u r t h e r and r e v e a l the presence o f other a r s e n i c c o n t a i n i n g f r a c t i o n s i n c l u d i n g t r i m e t h y l a r s i n e oxide. Thus, p r o v i d i n g SAM i s added, a l l the arsenic(V) intermediate i n Challenger's scheme can be i d e n t i f i e d i n c e l l e x t r a c t s of an organism which i s known to produce t r i m e t h y l a r s i n e . 11+

1I

3

2

2

2

7I+



7.

MCBRIDE E T

Alkylation of Arsenic

AL.

109

The r e s u l t of experiment 15, Table V I I i s p a r t i c u l a r l y i n t e r e s t i n g s i n c e , when e t h i o n i n e i s added i n place of methionine, no a r s i n e s are produced. The absence of e t h y l a r s i n e s argues a g a i n s t a p u r e l y chemical t r a n s f e r of an a l k y l group from sulphur to a r s e n i c , and the absence o f t r i m e t h y l a r s i n e argues s t r o n g l y f o r a methionine based enzyme i n v o l v e d s y n t h e t i c path s i n c e e t h i o n i n e i s a w e l l known antagonist to methionine (31). Along the same l i n e s i t has been found (.2,25) that phosphates i n h i b i t s the f o r mation of t r i m e t h y l a r s i n e from arsenate, a r s e n i t e , and methylarsonate, but not from cacodylate, by growing c u l t u r e s of C. humicola. The same organism can be p r e c o n d i t i o n e d by cacodyl a t e to produce t r i m e t h y l a r s i n e at a g r e a t e r than u s u a l r a t e from both arsenate and cacodylate (32). This i s seen i n F i g . 8. On the other hand,using the same organism, p r e c o n d i t i o n i n g w i t h arsenate r e s u l t s i n a dramatic r e d u c t i o n i n t r i m e t h y l a r s i n e production from cacodylate w h i l e i t o n l y s l i g h t l y s t i m u l a t e s production from arsenate. Model Studies Each step i n Challenger's mechanism i s c h e m i c a l l y reasona b l e . Indeed the a l k y l a t i o n steps are r e l a t e d to the w e l l known Meyer r e a c t i o n (20,32), which uses methyl i o d i d e or d i m e t h y l sulphate as the source of C H 3 + . Since SAM i s a sulphonium compound (34) other simpler analogues have been s t u d i e d w i t h respect to t h e i r a b i l i t y to methylate a r s e n i c (35). At pH 12 and 80°C (CH ) P"hPF ~ can be used f o r a l l the steps i n Challenger's scheme which i n v o l v e C H (these r e a c t i o n s can be monitored by NMR t e c h niques s i n c e the r e a c t a n t and products have d i s t i n c t chemical s h i f t s ) and each a r s e n i c ( V ) compound i n the scheme can be reduced to the a p p r o p r i a t e a r s e n i c ( I I I ) d e r i v a t i v e using S 0 . Thus the whole sequence can be e a s i l y d u p l i c a t e d . Methyl methionine i s o f t e n invoked as a model f o r SAM (34) and t h i s compound s l o w l y but i n c o m p l e t e l y , methylates CH AsO^~ at 25°C and a t the more r e a l i s t i c pH of 5.8. However a methyl sulphonium d e r i v a t i v e of CH -CoM under the same c o n d i t i o n s f a i l e d to t r a n s f e r i t s methyl group. 3

3

6

+

3

2

3

3

A r s e n i c Cycle i n the Environment Studies w i t h aerobic and anaerobic organisms have shown t h a t the former produce t r i m e t h y l a r s i n e whereas the l a t t e r produce the more c h e m i c a l l y r e a c t i v e d i m e t h y l a r s i n e . Taking these observat i o n s i n t o account, i t i s p o s s i b l e to p o s t u l a t e t h a t there i s an a r s e n i c c y c l e i n nature, a c y c l e that r e l i e s upon both b i o l o g i c a l and a b i o t i c r e a c t i o n s . Such a scheme i s o u t l i n e d i n F i g . 9. I t i s important not to view the r e a c t i o n s as being confined to sediment, water, or a i r but r a t h e r to ecosystems which are e i t h e r a e r o b i c or anaerobic because i t i s the a v a i l a b i l i t y of oxygen which w i l l determine the nature of the m i c r o b i a l f l o r a and w i l l i n f l u e n c e the f a t e and subsequent movement of the a r s i n e . Arsenate, a r s e n i t e , and methylarsonate r e a c t i n a s i m i l a r manner


ORGANOMETALS

AND

ORGANOMETALLOIDS

1400


1300 1000 ftoo

ο 4c «oo 400

1

Λ

200

0

/V'

10

30

20

AJ 40

50

4

60

FRACTION

Figure 7. Ion-exchange separation of C. humicola extracts incu bated with As-Na HAsOi, SAM, and NADPH. Peaks 1-4 are arsenite, cacodylic acid, methylarsonic acid, and arsenate, respec tively. 74

2

(CH ) As(nmote) 3 3

400"

Figure 8. Amount of (CH ) As in the headspace above growing 300· cultures of C. humicola precondi tioned in cacodylate (C and D) and not preconditioned (A and B). In Curves A and C the arsenical substrate is arsenate, in Β and D 0 it is cacodylate. s

s



7.

MCBRIDE E TA L .


ARSENIC

111

CYCLE

(

C H

a) * 3

,

"•(CH ) AiO(OH) 3

^

A»0(OH^=Â.(OH)3—•CH,>UO(OH) =*(CHÂ.io(OH)—i a

2

I •(CH,)Â«H (CH,)i.-S^X

ί Figure 9. Biological arsenic cycle. (=) aerobic or anaerobic; (· - -) aerobic biotic or abiotic; ( ) anaerobic; ( ) aerobic; (++) these reactions are prob ably abiotic.


112

ORGANOMETALS

AND ORGANOMETALLOIDS


i n both a e r o b i c and anerobic ecosystem. Cacodylate i s an important branch p o i n t ; aerobes reduce and methylate t h i s compound t o form t r i m e t h y l a r s i n e , anerobes reduce i t t o d i m e t h y l a r s i n e . The t r i m e t h y l a r s i n e i s e v e n t u a l l y o x i d i z e d t o cacodylate which can be i n c o r p o r a t e d d i r e c t l y i n t o the c y c l e o r f u r t h e r m o d i f i e d t o methylarsonate o r arsenate by m i c r o b i a l a c t i v i t y . D i m e t h y l a r s i n e can be o x i d i z e d t o cacodylate but i t may r e a c t w i t h other chemical c o n s t i t u e n t s i n i t s environment and thus i n i t i a t e an e n t i r e l y new s e t o f r e a c t i o n s . The l a t t e r i s a reasonable hypothesis when one considers the r e a c t i v i t y o f the a r s i n e and the e n v i r o n ment i n which i t i s produced. Acknowledgements T h i s paper i s a c o n t r i b u t i o n from the B i o i n o r g a n i c Chemistry Group supported i n p a r t by o p e r a t i n g and n e g o t i a t e d development grants from N.R.C. andM.R.C, Canada. BIBLIOGRAPHY 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

C h a l l e n g e r , F. (1945). Chem. Rev., 36, 315. Cox, D.P. and Alexander, M. (1973). A p p l i e d M i c r o . 25, 408. McBride, B.C. and Wolfe, R.S. (1971). B i o c h e m i s t r y , 10, 4312. Wolfe, R.S. (1971). Adv. in Microb. P h y s i o l . 6, 107. Stadtman, T.C. (1967). Ann. Rev. Microbiol. 21, 121. Barker, H.A. (1956). " B a c t e r i a l Fermentations", P. 1 John Wiley and Sons, I n c . , New York. McBride, B.C. and Wolfe, R.S. (1971). Adv. in Chem. S e r . 105, 11. Zeikus, J.G. (1977). B a c t e r i o l . Rev. 41, 514. B l a y l o c k , B.A. and Stadtman, T.C. (1966). Biochem. Biophys. Res. Commun. 11, 34. W o l i n , M.J., Wolin, E.A. and Wolfe, R.S. (1963). Biochem. Biophys. Res. Commun. 12, 465. B l a y l o c k , B.A. (1968). Archs. Biochem. Biophys. 124, 314. Wood, J.M. and Wolfe, R.S. (1966). Biochemistry 5, 3598. Roberton, A.M. and Wolfe, R.S. (1969). Biochim. Biophys. A c t a . 192, 420. Bryant, M.P., Wohn, E.A., Wolin, M.J. and Wolfe, R.S. (1967). Arch. M i k r o b i o l . 59, 20. McBride, B.C. (1970). D i s s e r t a t i o n . U n i v e r s i t y o f Illinois, Urbana, Illinois. McBride, B.C. and Wolfe, R.S. (1971). Biochemistry 10, 2317. T a y l o r , C.P. and Wolfe, R.S. (1974). J. Biol. Chem. 249, 4879. Wood, J.M., Kennedy, F.S. and Rosen, C.G. (1968). Nature 220, 173. Wolin, E.A., Wolin, M.J. and Wolfe, R.S. (1963). J. Biol. Chem. 238, 2882.


7.

20.

21. 22. 23. 24. 25.


26. 27. 28. 29. 30. 31. 32. 33. 34.

35.

MCBRIDE

ET

AL.


113

R a i z i s s , G.W. and Gavron, J.L. (1923). Organic a r s e n i c a l compounds, New York, N.Y. The Chemical Catalog Co., pp. 38-49. Smith, P.H. and Mah, R.A. (1966). A p p l . M i c r o . 14, 368. Bauchop, T. (1967). J . Bact. 94, 171. Peoples, S.A. (1975). A r s e n i c a l P e s t i c i d e s A.C.S. Symposium S e r i e s #7, p. 1. A v e l i n o , N., C u l l e n , W.R., McBride, B.C. Unpublished results. Woolson, E.A., Ed. (1975). A r s e n i c a l P e s t i c i d e s A.C.S. Symposium S e r i e s #7, Washington, D.C. Woolson, E.A. and Kearney, P.C. (1973). E n v i r o n . Sci. Technol. 7, 47. Isensee, A.R., Kearney, P.C., Woolson, E.A., Jones, G.E. and W i l l i a m s , V.P. (1973). E n v i r o n . Sci. Technol. 7, 841. C u l l e n , W.R., Froese, C L . , Lui, Α., McBride, B.C., Patmore, D.J. and Reimer, M. (1977). J. Organometal. Chem. 139, 61. Zingaro, R.A. and Irgolic, K . J . (1975). Science 187, 7651. C u l l e n , W.R., McBride, B.C. and Pickett, A.W. Unpublished results. Simmonds, S., Keller, E.B., Chandler, J.P. and duVigneaud, V. (1950). J. Biol. Chem. 183, 191. C u l l e n , W.R., McBride, B.C. and Reimer, M. Bull. E n v i r o n . Contam. T o x i c o l . , in p r e s s . Quick, A.J. and Adams, R. (1922). J . Amer. Chem. Soc. 44, 805. S a l v a t o r e , F. Borek, E., Zappia, V., Williams-Ashman, H.G., Schlenk, F., Eds. (1977). The Biochemistry o f Adenosyl methionine, Columbia U n i v e r s i t y P r e s s , New York, 1977. Chopra, A.K., C u l l e n , W.R., D o l p h i n , D. Unpublished r e s u l t s .

Discussion J . M. WOOD ( U n i v e r s i t y of Minnesota): I want t o ask t h i s question on b e h a l f of P r o f e s s o r C h a l l e n g e r , because i n h i s review a r t i c l e he a l l u d e s t o the p o s s i b l e r o l e o f coenzyme M i n methyl a t i o n of a r s e n i c by Methanobacteria. He has a s p e c u l a t i v e p a r a graph i n which he suggests t h a t the dimethylsulphonium d e r i v a t i v e would be a very good candidate f o r m e t h y l a t i o n of a r s e n i c . The methyl coenzyme M i t s e l f i s a bad candidate. Have you any i d e a whether h i s suggestion i s a good one? McBRIDE: enzyme M.

We haven't t r i e d any other compound on methyl c o

W. R. CULLEN ( U n i v e r s i t y of B r i t i s h Columbia): I n model s y s tems, t h a t methyl t r a n s f e r does take p l a c e from the methyl coen zyme M t o a r s e n i c ( I I I ) . Any sulphonium compound w i t h a methyl transfers to arsenic.


114

ORGANOMETALS

AND

ORGANOMETALLOIDS

G. Ε. ΡARRIS (Food and Drug A d m i n i s t r a t i o n ) : Do you have k i n e t i c data f o r t h i s t r a n s f e r that you are t a l k i n g about?

this.

CULLEN: Yes, we have attempted t o measure some k i n e t i c s on I t ' s a v e r y d i f f i c u l t system t o work on.


M. 0. ANDREAE ( S c r i p p s I n s t i t u t e of Oceanography): Do you have evidence f o r the formation of l a r g e r compounds ; would you see them i f they were formed? McBRIDE:

By l a r g e r compounds do you mean complexes?

ANDREAE:

Yes, arseno-betaine or something l i k e t h a t .

McBRIDE: We don't have any good evidence, but r e c e n t l y we ran a Sephadex column, a g e l f i l t r a t i o n column, and found a r a d i o a c t i v e f r a c t i o n t h a t i n d i c a t e s a h i g h molecular weight. ANDREAE: D i d you do a mass balance t h a t w i l l t e l l you how much of the l a b e l can be accounted f o r by arsenate and other com pounds? McBRIDE:

Yes, we can account f o r 100%.

ANDREAE: In your experiments w i t h the marine mud, were you u s i n g a rubber t r a p t o assay f o r the a r s i n e or was t h a t determined by head space a n a l y s i s ? McBRIDE:

No, there was a t r a p .

F. E. BRINCKMAN ( N a t i o n a l Bureau of Standards): I'm i n t r i g u e d by t h i s apparent complexation on the Dowex column. I s i t engendered by the b i o a c t i v i t y ? That i s , might there be a t h i r d component t h a t leads t o complexation of these a r s e n i c species? [McBRIDE: Yes] T y p i c a l l y , under the c o n d i t i o n s you use f o r e l u t i o n , these are an i o n i c species from the pKa v a l u e s . In our l a b o r a t o r y , f o r HPLC, we use both weak and s t r o n g anion and c a t i o n columns and can make s a t i s f a c t o r y s e p a r a t i o n s of demethylated me t a b o l i t e s from s o i l b a c t e r i a i n v o l v i n g the m e t h y l a r s e n i c a l p e s t i c i d e s . We see no evidence of t h i s k i n d of complexation. We are concerned about higher molecular weight e l u a n t s because of your work and t h a t of P r o f e s s o r I r g o l i c concerning the b e t a i n e or other p o s s i b l e a n i o n i c species w i t h a l a r g e molecule pendant. McBRIDE: I t seems t o be r e a l , and i t seems to be a s s o c i a t e d w i t h a b i o l o g i c a l l y a c t i v e e x t r a c t . I f you i n a c t i v a t e the e x t r a c t you don't see these. You can use l a b e l s (C-14 c a c o d y l , C-14 meth y l a r s o n i c a c i d , As-74 arsenate) and you do not see these complexes form. E v e r y t h i n g e l u t e s at the a p p r o p r i a t e p o i n t , but when you have a b i o l o g i c a l l y a c t i v e system, then you see t h i s change t o


7.

MCBRIDE E T A L .

Alkyhtion of Arsenic

115

what we t h i n k i s a complex formation. I t ' s very dramatic when you look at C-14 cacodyl as a s u b s t r a t e r a t h e r than as arsenate. A l most 99% of the m a t e r i a l moves i n t o a f r a c t i o n which e l u t e s w i t h water.


BRINCKMAN: This i s a very c l e a r demonstration that c a u t i o n should be e x e r c i s e d i n s p e c i a t i n g these t r a c e m a t e r i a l s , metabol i t e s p a r t i c u l a r l y , when u s i n g methodology l i k e Braman's technique of r e d u c t i v e v o l a t i l i z a t i o n to form h y d r i d e s . You may l o s e a r a t h e r c r i t i c a l molecule which i s important f o r c o u p l i n g s e v e r a l i o n i c species and which may be i n d i c a t i v e of the mechanistic pathway. K. J . IRGOLIC (Texas A & M u n i v e r s i t y ) : You a l l u d e to the p o s s i b l e transformation of t r i m e t h y l a r s i n e to d i m e t h y l a r s i n i c a c i d i n some of your systems. In t e s t tube r e a c t i o n s under aerobic c o n d i t i o n s , i t ' s a r e l a t i v e l y slow r e a c t i o n , but you break down the compound, l o s e one methyl group, and end up w i t h d i a l k y l a r s i n i c s from t r i a l k y l a r s i n e s . ANDREAE: Your marine mud produces h a r d l y any methane. Where d i d you get t h a t mud? Some marine muds produce methane, some do not. McBRIDE: We d i d n ' t f i n d any marine mud that was producing l o t of methane although o b v i o u s l y they e x i s t . ANDREAE: i n the core?

Was

your mud

a

sample from a r e l a t i v e l y shallow l e v e l

McBRIDE: No, we went f a i r l y deep. There was a l o t of s u l f u r i n these muds, whether t h a t was i n f l u e n c i n g what was going on, I don't know. RECEIVED

August

22,

1978.


Organometals and Organometalloids - American Chemical Society

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