Hernopexin (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 O-2-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: [GRAPHICS] values of k(on), k(off), and K (=k(on)/k(off)) are (6.3 +/- 0.3) x 10(3) M-1 s(-1), (9.1 +/- 0.4) x 10(-4) s(-1), and (6.9 +/- 0.6) x 10(6) M-1, respectively, at pH 7.0 and 10.0 degrees C. O-2 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 + O-2 reversible arrow(hon) HPX-heme(III)-N(O)OO ->(l) HPX-heme(III) + NO3-the backward reaction rate is negligible. Values of h., and I are (2.4 +/- 0.3) x 10 1 M-1 s(-1) and (1.4 +/- 0.2) x 10(-3) s(-1), respectively, at pH 7.0 and 10.0 degrees 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 (gimel(max) = 409 nm and epsilon(409) = 1.51 x 10(5) M-1 cm(-1)). These results, representing the first kinetic evidence for HPX-heme(II) nitrosylation and O-2-mediated oxidation of HPX-heme(II)-NO, might be predictive of transient (pseudo-enzymatic) function(s) of heme carriers. (c) 2006 Elsevier Inc. All rights reserved.