Nitric oxide (NO) has been found to inhibit the actions of the transmembrane metal reductase Fre1 in the yeast Saccharomyces cerevisiae. This membrane-spanning heme protein is homologous to the gp91^PHOX protein of the NADPH oxidase enzyme complex and is responsible for reducing extracellular oxidized metals (i.e., ferric and cupric ions) before high-affinity uptake. Consistent with its role in metal metabolism, inhibition of Fre1 by NO also inhibited yeast growth in low-iron medium. Inhibition by NO was found to be O₂-dependent and irreversible. Further examination of the chemistry responsible for activity loss shows that the generation of N₂O₃ via NO–O₂ chemistry was responsible for the activity loss, possibly via nitrosation of the protein followed by loss of the heme prosthetic group.