Scheme 8. Comparison of Oxygen Incorporation during CAM Formation via Different Pathways.

Although pathway III better describes the native CmlI pathway, the off-cycle or artificial reaction of P with NH(OH)-CAM was carried out to make a direct comparison to the analogous reaction in the AurF cycle. In accord with the results reported for the anaerobic reaction of the AurF-peroxo intermediate reaction with NH(OH)-Ar,¹³ we find that CmlI^red and CAM are produced when P is reacted with NH(OH)-CAM. This reaction would not be on-pathway for Scheme 8, pathway III, as only CmlI^ox and NH(OH)-CAM are found together in the active site, thereby allowing the redox reaction to occur. Consequently, the off-pathway reaction for CmlI might occur as proposed for AurF (Scheme 8, pathway II). However, the subsequent addition of O₂ was found to yield additional CAM and CmlI finishes in the mostly oxidized rather than reduced or P states expected based on the reported AurF reaction. ¹³ Additional kinetic and spectroscopic studies will be required to fully characterize the off-pathway reaction of P with NH(OH)-CAM.