Bacterial nitric-oxide reductase catalyzes the two electron reduction of nitric oxide to nitrous oxide. In the oxidized form the active site non-heme FeB and high spin heme b 3 are µ-oxo bridged. The hemeb 3 has a ligand-to-metal charge transfer band centered at 595 nm, which is insensitive to pH over the range of 6.0–8.5. Partial reduction of nitric-oxide reductase yields a three electron-reduced state where only the hemeb 3 remains oxidized. This results in a shift of the heme b 3 charge transfer band ?max to longer wavelengths. At pH 6.0 the charge transfer band ?max is 605 nm, whereas at pH 8.5 it is 635 nm. At pH 6.5 and 7.5 the nitric-oxide reductase ferric hemeb 3 population is a mixture of both 605- and 635-nm forms. Magnetic circular dichroism spectroscopy suggests that at all pH values examined the proximal ligand to the ferric hemeb 3 in the three electron-reduced form is histidine. At pH 8.5 the distal ligand is hydroxide, whereas at pH 6.0, when the enzyme is most active, it is water.