Reductive nitrosylation of ferric microperoxidase-11

Abstract

Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c, which is considered as a heme-protein model. Here, the reductive nitrosylation of ferric MP11 (MP11(III)) under anaerobic conditions has been investigated between pH 7.4 and 9.2, at T = 20.0 °C. At pH ≤ 7.7, NO binds reversibly to MP11(III) leading to the formation of the MP11(III)–NO complex. However, between pH 8.2 and 9.2, the addition of NO to MP11(III) leads to the formation of ferrous nitrosylated MP11(II) (MP11(II)–NO). In fact, the transient MP11{FeNO}⁶ species is converted to ferrous deoxygenated MP11 (MP11(II)) by OH⁻- and H₂O-based catalysis, which represents the rate-limiting step of the whole reaction. Then, MP11(II) binds NO very rapidly leading to MP11(II)–NO formation. Over the whole pH range explored, the apparent values of k_on, k_off, and K (= k_off/k_on) for MP11(III)(–NO) (de)nitrosylation are essentially pH independent, ranging between 5.8 × 10⁵ M⁻¹ s⁻¹ and 1.6 × 10⁶ M⁻¹ s⁻¹, between 1.9 s⁻¹ and 3.7 s⁻¹, and between 1.4 × 10⁻⁶ M and 4.6 × 10⁻⁶ M, respectively. Values of the apparent pseudo-first-order rate constant for the MP11{FeNO}⁶ conversion to MP11(II) (i.e., h) increase linearly with pH; the apparent values $h_{\text{OH}^{-}}$ and $h_{{{\text{H}}_{ 2} {\text{O}}}}$ are 7.2 × 10² M⁻¹ s⁻¹ and 2.5 × 10⁻⁴ s⁻¹, respectively. Present data confirm that MP11 is a useful molecular model to highlight the role of the protein matrix on the heme-based reactivity.