Reductive nitrosylation and peroxynitrite‐mediated oxidation of heme–hemopexin

Hemopexin (HPX), which serves as a scavenger and transporter of toxic plasma heme, has been postulated to play a key role in the homeostasis of NO. In fact, HPX–heme(II) reversibly binds NO and facilitates NO scavenging by O 2 . HPX–heme is formed by two four‐bladed β‐propeller domains. The heme is bound between the two β‐propeller domains, residues His213 and His266 coordinate the heme iron atom. HPX–heme displays structural features of heme‐proteins endowed with (pseudo‐)enzymatic activities. In this study, the kinetics of rabbit HPX–heme(III) reductive nitrosylation and peroxynitrite‐mediated oxidation of HPX–heme(II)–NO are reported. In the presence of excess NO, HPX–heme(III) is converted to HPX–heme(II)–NO by reductive nitrosylation. The second‐order rate constant for HPX–heme(III) reductive nitrosylation is (1.3 ± 0.1) × 10 1   m −1 ·s −1 , at pH 7.0 and 10.0 °C. NO binding to HPX–heme(III) is rate limiting. In the absence and presence of CO 2 (1.2 × 10 −3   m ), excess peroxynitrite reacts with HPX–heme(II)–NO (2.6 × 10 −6   m ) leading to HPX–heme(III) and NO, via the transient HPX–heme(III)–NO species. Values of the second‐order rate constant for HPX–heme(III)–NO formation are (8.6 ± 0.8) × 10 4 and (1.2 ± 0.2) × 10 6   m −1 ·s −1 in the absence and presence of CO 2 , respectively, at pH 7.0 and 10.0 °C. The CO 2 ‐independent value of the first‐order rate constant for HPX–heme(III)–NO denitrosylation is (4.3 ± 0.4) × 10 −1  s −1 , at pH 7.0 and 10.0 °C. HPX–heme(III)–NO denitrosylation is rate limiting. HPX–heme(II)–NO appears to act as an efficient scavenger of peroxynitrite and of strong oxidants and nitrating species following the reaction of peroxynitrite with CO 2 (e.g. ONOOC(O)O – , CO 3 – , and NO 2 ).