Fe(B)Mbs are structural and functional models of native bacterial nitric oxide reductases (NORs) generated through engineering of myoglobin. These biosynthetic models replicate the heme-nonheme diiron site of NORs and allow substitutions of metal centers and heme cofactors. Here, we provide evidence for multiple NOR turnover in monoformyl-heme-containing Fe(B)Mb1 proteins loaded with Fe-II, Co-II, or Zn-II metal ions at the Fe-B site (Fe-II/Co-II/Zn-II-Fe(B)Mb 1 (MF-heme)). FTIR detection of the nu(NNO) band of N2O at 2231 cm(-1) provides a direct quantitative measurement of the product in solution. A maximum number of turnover is observed with Fe-II-Fe(B)Mb1 (MF-heme), but the NOR activity is retained when the Fe-B site is loaded with Zn-II. These data support the viability of a one-electron semireduced pathway for the reduction of NO at binuclear centers in reducing conditions.