title: Direct observation of the low-spin Fe(III)-NO(radical) intermediate state during rebinding of NO to photodeligated ferric cytochrome c.
abstract: Nitrosylated ferric heme is autoreduced readily to the more stable Fe(II)-NO adduct, but it is stabilized in NO-carrier heme proteins where maintaining the Fe(III) oxidation state is crucial for efficient NO delivery. Density functional theory calculations by Lehnert and co-workers have shown that a NO-bound ferric model heme has a low-spin (LS) Fe(III)-NO(radical) state that might be critical for efficient NO transport by NO-carrier heme proteins. Recently, the elusive LS Fe(III)-NO(radical) state was observed as an electronic intermediate state during geminate rebinding (GR) of NO to ferric myoglobin (Mb(III)). Cytochrome c (Cytc), a ubiquitous heme protein, is useful for generalizing the presence of the LS Fe(III)-NO(radical) state. Photoexcitation dynamics of NO-bound ferric Cytc (Cytc(III)NO) was probed after excitation of Cytc(III)NO in D2O solution at 294 K with a 575 nm pulse using femtosecond vibrational spectroscopy. The time-resolved spectra displayed several weak absorption bands in the 1900-1800 cm(-1) range and a dominant bleach at 1917 cm(-1), the position of the absorption at equilibrium. Two absorptions, with 37 cm(-1) isotope shift of (15)NO, shifted toward higher energy and narrowed with an average time constant of 8 ps, indicating that they arose from thermally and/or vibrationally excited NO in the ground electronic state of Cytc(III)NO. Three absorption bands, showing 33 cm(-1) isotope shift of (15)NO and peaked at 1865, 1836, and 1807 cm(-1), were assigned to the deligated NO residing in the interior of the protein, to the rebound Cytc(III)NO in the LS Fe(III)-NO(radical) state, and to the vibrationally excited NO of Cytc(III)NO in the LS Fe(III)-NO(radical) state, respectively. The quantum yield for NO deligation of Cytc(III)NO by a 575 nm photon was 0.8 ± 0.1. Most of the deligated NO showed non-exponential GR, and the GR kinetics was described by exp(-(t/7 ps)(0.7)). Every rebound Cytc(III)NO formed the LS Fe(III)-NO(radical) state that relaxed into the ground state, with the relaxation kinetics described by exp(-(t/2.5 ps)(0.7)). The GR of NO to ferric Cytc was as fast as the thermal relaxation of hot heme, and the relaxation of the rebound Cytc(III)NO in the intermediate LS Fe(III)-NO(radical) state was faster than the thermal relaxation of hot heme, generating the rebound Cytc(III)NO in a thermally excited ground electronic state. For both Cytc(III)NO and Mb(III)NO, the relaxation rate of the LS Fe(III)-NO(radical) state was similar to the upper rate limit of the domed-to-planar heme transition observed in NO-rebound ferrous-heme proteins, suggesting that the change in the Fe-NO bond length is coupled to the doming motion of the heme Fe.
authors.affiliation: Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University , Busan 609-735, Korea.
authors.forename: Jaeheung
authors.initials: J
authors.lastname: Park
authors.forename: Taegon
authors.initials: T
authors.lastname: Lee
authors.forename: Manho
authors.initials: M
authors.lastname: Lim
mesh_terms: D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism*; D017666:Deuterium Oxide / Q000737:chemistry; D005290:Ferric Compounds / Q000737:chemistry*; D007700:Kinetics; D008027:Light; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D009587:Nitrogen Isotopes / Q000737:chemistry; D011789:Quantum Theory; D013055:Spectrophotometry, Infrared; D013696:Temperature
chemical_list: D005290:Ferric Compounds; D009587:Nitrogen Isotopes; D009569:Nitric Oxide; D045304:Cytochromes c; D017666:Deuterium Oxide