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 O-2. Removal of distal pocket steric hindrance via a Leu -> Ala mutation yields favorable O-2 binding (K-d similar to 49 nM) without apparent H-bond stabilization of the Fe-O-2 moiety, as well as an extremely high distal heme-NO affinity (K-d similar to 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.