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.