A Distal Pocket Leu Residue Inhibits the Binding of O<sub>2</sub> and NO at the Distal Heme Site of Cytochrome <i>c</i>′ GartonElizabeth M. PixtonDavid A. PetersenChristine A. EadyRobert R. HasnainS. Samar AndrewColin R. 2016 Cytochromes <i>c</i>′ are pentacoordinate heme proteins with sterically hindered distal sites that bind NO and CO but do not form stable complexes with O<sub>2</sub>. Removal of distal pocket steric hindrance via a Leu→Ala mutation yields favorable O<sub>2</sub> binding (<i>K</i><sub>d</sub> ∼49 nM) without apparent H-bond stabilization of the Fe–O<sub>2</sub> moiety, as well as an extremely high distal heme-NO affinity (<i>K</i><sub>d</sub> ∼70 fM). The native Leu residue inhibits distal coordination of diatomic ligands by decreasing <i>k</i><sub>on</sub> as well as increasing <i>k</i><sub>off</sub>. The connection between distal steric constraints, <i>k</i><sub>off</sub> values, and distal to proximal heme-NO conversion is discussed.