The tetraheme cytochrome c(554) is the redox partner of hydroxylamine oxidoreductase (HAO) of Nitrosomonas europaea, responsible for accepting the four electrons generated in HAO turnover. Here, we have used protein film voltammetry (PFV) to provide a new, highly resolved picture of the redox properties of cyt c(554). The four heme groups of the cytochrome are found to have potentials of +32, +50, -183, and -283 mV (versus hydrogen). While the two lowest potential hemes (III and IV) behave as simple n(app) = 1 redox cofactors in all experiments, the high potential hemes appear to act as a pair that engages in cooperative electron transfer reactions (n(app) > 1.0). By studying the cytochrome on variable electrode surfaces, gating of the reduction of the two high potential hemes can also be observed. When the interfacial electron transfer rate for cyt c(554) is on the time scale of HAO catalysis (on a mercaptoundecanoic acid-modified gold electrode), gating is found, indicating that oxidation of the two-electron reduced form of cyt c(554) is prevented, while reduction of the totally oxidized form is facile.