O2-mediated oxidation of hemopexin-heme(II)-NO

Hemopexin (HPX), serving as scavenger and transporter of toxic plasma heme, has been postulated to play a key role in the homeostasis of NO. Here, kinetics of HPX-heme(II) nitrosylation and O₂-mediated oxidation of HPX-heme(II)-NO are reported. NO reacts reversibly with HPX-heme(II) yielding HPX-heme(II)-NO, according to the minimum reaction scheme:{A formula is presented}values of k_on, k_off, and K (=k_on/k_off) are (6.3 ± 0.3) × 10³ M^-1 s^-1, (9.1 ± 0.4) × 10^-4 s^-1, and (6.9 ± 0.6) × 10⁶ M^-1, respectively, at pH 7.0 and 10.0 °C. O₂ reacts with HPX-heme(II)-NO yielding HPX-heme(III) and multiscripts(NO, mml:none(), mml:none(), 3, -), by means of the ferric heme-bound peroxynitrite intermediate (HPX-heme(III)-N(O)OO), according to the minimum reaction scheme:{A formula is presented}the backward reaction rate is negligible. Values of h_on and l are (2.4 ± 0.3) × 10¹ M^-1 s^-1 and (1.4 ± 0.2) × 10^-3 s^-1, respectively, at pH 7.0 and 10.0 °C. The decay of HPX-heme(III)-N(O)OO (i.e., l) is rate limiting. The HPX-heme(III)-N(O)OO intermediate has been characterized by optical absorption spectroscopy in the Soret region (λ_max = 409 nm and ε₄₀₉ = 1.51 × 10⁵ M^-1 cm^-1). These results, representing the first kinetic evidence for HPX-heme(II) nitrosylation and O₂-mediated oxidation of HPX-heme(II)-NO, might be predictive of transient (pseudo-enzymatic) function(s) of heme carriers.