The wood preservative chromated copper arsenate is a substrate for trimethylarsine biosynthesis.

The wood preservative chromated copper arsenate (CCA) is a very widely used product. As it contains both copper and arsenic, it is not dissimilar to the pigments Scheele's green and Schweinfurter's green which were found to be biologically convertible to the toxic Gosio gas (B. Gosio, Ber. 30:1024-1026, 1897) later identified by Challenger and co-workers as trimethylarsine (F. Challenger, Adv. Enzymol. 12:429-491, 1951). Thus, it was of interest to determine whether microbiological action on CCA and wood treated with CCA could result in the production of trimethylarsine. We report that the fungus Candida humicola will produce this arsine from dilute solutions of CCA and from wood soaked in CCA.

The reduction of trimethylarsine oxide by Candida humicola.

Trimethylarsine oxide, a probable intermediate in the biological transformation of arsenate, was reduced to volatile trimethylarsine by Candida humicola. A simple assay for the rate of trimethylarsine production from trimethylarsine oxide by the fungus was developed. The optimum pH for the reduction was determined as 5.1-5.2, and the optimum temperature was 40 degrees C. The rate of reduction was directly proportional to cell concentration and followed Michaelis-Menten type kinetics. There was almost no trimethylarsine produced by heated or broken cells. The reaction was inhibited by a number of electron transport inhibitors and uncouplers of oxidative phosphorylation including cyanide, azide, and 2,4-dinitrophenol. The rate of reduction was modified by arsenate, methylarsonate, dimethylarsinate, selenate, and tellurate. Preincubation of cells with trimethylarsine oxide increased the rate of reduction 69-fold; this increase in activity was blocked if the cells were incubated with the protein synthesis inhibitor cycloheximide.

The transformation of arsenicals by Candida humicola.

An analytical procedure for the separation, detection, and identification of some of the compounds produced by a cell preparation of the fungus Candida humicola from 74As-arsenate, [14C]-methylarsonate, and [14C]-dimethylarsinate has been devised and tested. It has been possible to detect five distinct 74As-containing compounds following the incubation of 74As-arsenate with a broken cell homogenate, three of which have been identified as arsenite, methylarsonate, and dimethylarsinate. With [14C]methylarsonate as substrate, [14C]dimethylarsinate and [14C]trimethylarsine oxide are produced. Products from [14C]dimethylarsinate include [14C]methylarsonate and [14C]trimethylarsine oxide as well as an unknown species. The absence of any transformation when buffer replaces the cell preparation implicates these various compounds as intermediates in a biological synthesis of trimethylarsine.

Effect of phosphate and other anions on trimethylarsine formation by Candida humicola.

Phosphate inhibited the formation of trimethylarsine from arsenite, arsenate, and monomethylarsonate, but not from dimethylarsinate, by growing cultures of Candida humicola. Phosphite suppressed trimethylarsine production by growing cultures from monomethylarsonate but not from arsenate and dimethylarsinate, and hypophosphite caused a temporary inhibition of both proliferation and the conversion of these three arsenic sources to trimethylarsine. Resting cells of C. humicola derived from cultures grown in arsenic-free media generated the volatile arsenical only after a lag phase. High antimonate concentrations reduced the rate of conversion of arsenate to trimethylarsine by resting cells, but nitrate was without effect.