Resonance Raman studies of cytochrome c ' support the binding of NO and COto opposite sides of the heme: Implications for ligand discrimination in heme-based sensors

Resonance Raman (RR) studies have been conducted on Alcaligenes xylosoxidan s cytochrome c', a mono-His ligated hemoprotein which reversibly binds NO a nd CO but not O-2. Recent crystallographic characterization of this protein has revealed the first example of a hemoprotein which can utilize both sid es of its heme (distal and proximal) for binding exogenous ligands to its F e center. The present RR investigation of the Fe coordination and heme pock et environments of ferrous, carbonyl, and nitrosyl forms of cytochrome c' i n solution fully supports the structures determined by X-ray crystallograph y and offers insights into mechanisms of ligand discrimination in heme-base d sensors. Ferrous cytochrome c' reacts with CO to form a six-coordinate he me-CO complex, whereas reaction with NO results in cleavage of the proximal linkage to give a five-coordinate heme-NO adduct, despite the relatively h igh stretching frequency (231 cm(-1)) of the ferrous Fe-N(His) bond. RR spe ctra of the six-coordinate CO adduct indicate that CO binds to the Fe in a nonpolar environment in line with its location in the hydrophobic distal he me pocket. On the other hand, RR data for the five-coordinate NO adduct sug gest a positively polarized environment for the NO ligand, consistent with its binding close to Arg 124 on the opposite (proximal) side of the heme. P arallels between certain physicochemical properties of cytochrome c' and th ose of heme-based sensor proteins raise the possibility that the latter may also utilize both sides of their hemes to discriminate between NO and CO b inding.