REDOX BUFFERING BY MELANIN AND FE(II) IN CRYPTOCOCCUS-NEOFORMANS

Melanin is a fungal extracellular redox buffer which, in principle, ca n neutralize antimicrobial oxidants generated by immunologic effector cells, but its source of reducing equivalents is not known, We wondere d whether Fe(II) generated by the external ferric reductase of fungi m ight have the physiologic function of reducing fungal melanin and ther eby promoting pathogenesis, We observed that exposure of a melanin fil m electrode to reductants decreased the open-circuit potential (OCP) a nd reduced the area of a cyclic voltammetric reduction wave whereas ex posure to oxidants produced the opposite effects, Exposure to 10, 100, 1,000 or 10,000 mu M Fe(II) decreased the OCP of melanin by 0.015, 0. 038, 0.100, and 0.120 V, respectively, relative to a silver-silver chl oride standard, and decreased the area of the cyclic voltammetric redu ction wave by 27, 35, 50, and 83%, respectively, Moreover, exposure to Fe(II) increased the buffering capacity by 44%, while exposure to mil limolar dithionite did not increase the buffering capacity, The ratio of the amount of bound iron to the amount of the incremental increase in the following oxidation wave was approximately 1.0, suggesting that bound iron participates in buffering, Light absorption by melanin sus pensions was decreased 14% by treatment with Pe(EI), consistent with r eduction of melanin, Light absorption by suspensions of melanized Cryp tococcus neo-formans was decreased 1.3% by treatment with Fe(II) (P < 0.05), Cultures of C. neoformans generated between 2 and 160 mu M Fe(I I) in culture supernatant, depending upon the strain and the condition s [the higher values were achieved by a constitutive ferric reductase mutant in high concentrations of Fe(III)]. We infer that Fe(II) can re duce melanin under physiologic conditions; moreover, it binds to melan in and cooperatively increases redox buffering, The data support a mod el for physiologic redox cycling of fungal melanin, whereby electrons exported by the yeast to form extracellular Fe(II) maintain the reduci ng capacity of the extracellular redox buffer.