Accommodation of Two Diatomic Molecules in Cytochrome bo3: Insights into NO Reductase Activity in Terminal Oxidases

Bacterial heme-copper terminal oxidises 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 N2O. 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 N2O. The EPR and resonance Raman signatures of the hence o(3)-NO complex are comparable to chose 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 CUB-nitrosyl complex, but that instead, die 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 center dot OC-CUB] tertiary complex car 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 CUB is not an essential step of the reaction mechanism.