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 (K_d ∼49 nM) without apparent H-bond stabilization of the Fe–O₂ moiety, as well as an extremely high distal heme-NO affinity (K_d ∼70 fM). The native Leu residue inhibits distal coordination of diatomic ligands by decreasing k_on as well as increasing k_off. The connection between distal steric constraints, k_off values, and distal to proximal heme-NO conversion is discussed.