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:HPX-heme(II)+NO⇌koffkonHPX-heme(II)-NOvalues of kon, koff, and K (=kon/koff) 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 NO3-, by means of the ferric heme-bound peroxynitrite intermediate (HPX-heme(III)-N(O)OO), according to the minimum reaction scheme:HPX-heme(II)-NO+O2⇌honHPX-heme(III)-N(O)OO→lHPX-heme(III)+NO3-the backward reaction rate is negligible. Values of hon 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 (λmax = 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.