Picosecond Binding of the His Ligand to Four-Coordinate Heme in Cytochrome c′: A One-Way Gate for Releasing Proximal NO

We provide a direct demonstration of a "kinetic trap" mechanism in the proximal 5-coordinate heme-nitrosyl complex (5c-NO) of cytochrome cfrom Alcaligenes xylosoxidans (AXCP) in which picosecond rebinding of the endogenous His ligand following heme-NO dissociation acts as a one-way gate for the release of proximal NO into solution. This demonstration is based upon picosecond transient absorption changes following NO photodissociation of the proximal 5c-NO AXCP complex. We have determined the absolute transient absorption spectrum of 4-coordinate ferrous heme to which NO rebinds with a time constant τNO = 7 ps (kNO = 1.4 × 10 11 s −1 ) and shown that rebinding of the proximal histidine to the 4-coordinate heme takes place with a time constant τHis = 100 ± 10 ps (kHis =1 0 10 s −1 ) after the release of NO from the proximal heme pocket. This rapid His reattachment acts as a one-way gate for releasing proximal NO by precluding direct proximal NO rebinding once it has left the proximal heme pocket and requiring NO rebinding from solution to proceed via the distal heme face.