A Distal Pocket Leu Residue Inhibits the Binding of O2 and NO at the Distal Heme Site of Cytochrome c

Cytochromes c′ are pentacoordinate heme proteins with sterically hindered distal sites that bind NO and CO but do not form stable complexes with O2. Removal of distal pocket steric hindrance via a Leu→Ala mutation yields favorable O2 binding (Kd ∼49 nM) without apparent H-bond stabilization of the Fe–O2 moiety, as well as an extremely high distal heme-NO affinity (Kd ∼70 fM). The native Leu residue inhibits distal coordination of diatomic ligands by decreasing kon as well as increasing koff. The connection between distal steric constraints, koff values, and distal to proximal heme-NO conversion is discussed.