Accommodation of two diatomic molecules in cytochrome bo ₃: Insights into NO reductase activity in terminal oxidases

Bacterial heme-copper terminal oxidases react quickly with NO to form a heme - nitrosyl complex, which, in some of these enzymes, can further react with a second NO molecule to produce N ₂O. Previously, we characterized the heme a ₃-NO complex formed in cytochrome ba ₃ from Thermus thermophilus and the product of its low-temperature illumination. We showed that the photolyzed NO group binds to Cu _B(I) to form an end-on NO-Cu _B or a side-on copper-nitrosyl complex, which is likely to represent the binding characteristics of the second NO molecule at the heme-copper active site. Here we present a comparative study with cytochrome bo ₃ from Escherichia coli. Both terminal oxidases are shown to catalyze the same two-electron reduction of NO to N ₂O. The EPR and resonance Raman signatures of the heme o ₃-NO complex are comparable to those of the a ₃-NO complex. However, low-temperature FTIR experiments reveal that photolysis of the heme o ₃-NO complex does not produce a Cu _B-nitrosyl complex, but that instead, the NO remains unbound in the active-site cavity. Additional FTIR photolysis experiments on the heme-nitrosyl complexes of these terminal oxidases, in the presence of CO, demonstrate that an [o ₃-NO·OC-Cu _B] tertiary complex can form in bo ₃ but not in ba ₃. We assign these differences to a greater iron-copper distance in the reduced form of bo ₃ compared to that of ba ₃. Because this difference in metal-metal distance does not appear to affect the NO reductase activity, our results suggest that the coordination of the second NO to Cu _B is not an essential step of the reaction mechanism.