Is Saccharomyces cerevisiae azoreductase the plasma membrane ferric reductase?

Abstract

Unspecific bacterial reduction of azo dyes is a widely studied process in correlation with the biological treatment of coloured waste waters but the enzyme system associated to this bacterial capability has never been positively identified. Several ascomycete yeast strains, isolated by our group from contaminated soils, display similar decolourising activities. A study of the yeast-mediated process in batch culture demonstrated that colour loss was due to a reductase activity, expressed constitutively, which could transform azo dyes into colourless amines. In order to get a better understanding of the azoreductase activity of yeast cells we selected a Saccharomyces cerevisiae strain (BLC0276) with a high decolourising capacity. A likely candidate might be the plasma membrane ferric reductase system. Both azoreductase and ferric reductase have peak activities in the late exponential growth phase, and their substrates -ferricyanide and soluble azo dyes - are impermeant to the cell plasma membrane. This hypothesis was confirmed through two different lines of evidence: the use of different inhibitors and the construction of mutants defective in the iron reductase system. Inhibitors like carbonylcyanide m-chlorophenylhydrazone (CCCP), diphenylene iodonium (DPI), p-chloromercuribenzoate (pCMB) and chloroquine (CQ), were tested. In most cases the percentage inhibition of both activities was similar. The genes Fre1 and Fre2 were deleted and the effect on the decolourising activity was analysed in the mutant strains. The effect of Fre2 deletion was negligible, but fre1 and fre1fre2 strains showed a much reduced decolourising capability. These results demonstrate the involvement of the ferric reductase system in the azo decolourising capacity of S. cerevisiae. However the capacity was not completely removed indicating that cells have an alternative reducing system.