Nitric oxide reductases (NORs) reduce nitric oxide to nitrous oxide in the denitrification pathway of the global nitrogen cycle. NORs contain four iron cofactors and the NO reduction occurs at the heme b3/nonheme FeB binuclear active site. The determination of reduction potentials of the iron cofactors will help us elucidate the enzymatic reaction mechanism. However, previous reports on these potentials remain controversial. Herein, we performed electrochemical and surface-enhanced infrared absorption (SEIRA) spectroscopic measurements of Pseudomonas aeruginosa NOR immobilized on gold electrodes. Cyclic voltammograms exhibited two reduction peaks at -0.11 and -0.44 V vs SHE, and a SEIRA spectrum using a vibrational probe of CO showed a characteristic band at 1972 cm-1 at -0.4 V vs SHE, which was assigned to νCO of heme b3-CO. Our results suggest that the reduction of heme b3 initiates the enzymatic NO reduction.