Nitrosylation of ferric zebrafish nitrobindin: A spectroscopic, kinetic, and thermodynamic study

Nitrobindins (Nbs) are all-β-barrel heme-proteins present in all the living kingdoms. Nbs inactivate reactive nitrogen species by sequestering NO, converting NO to HNO₂, and isomerizing peroxynitrite to NO₃^- and NO₂^-. Here, the spectroscopic characterization of ferric Danio rerio Nb (Dr-Nb(III)) and NO scavenging through the reductive nitrosylation of the metal center are reported, both processes being relevant for the regulation of blood flow in fishes through poorly oxygenated tissues, such as retina. Both UV-Vis and resonance Raman spectroscopies indicate that Dr-Nb(III) is a mixture of a six-coordinated aquo- and a five-coordinated species, whose relative abundancies depend on pH. At pH ≤ 7.0, Dr-Nb(III) binds reversibly NO, whereas at pH ≥ 7.8 NO induces the conversion of Dr-Nb(III) to Dr-Nb(II)-NO. The conversion of Dr-Nb(III) to Dr-Nb(II)-NO is a monophasic process, suggesting that the formation of the transient Dr-Nb(III)-NO species is lost in the mixing time of the rapid-mixing stopped-flow apparatus (∼ 1.5 ms). The pseudo-first-order rate constant for the reductive nitrosylation of Dr-Nb(III) is not linearly dependent on the NO concentration but tends to level off. Values of the rate-limiting constant (i.e., k_lim) increase linearly with the OH^- concentration, indicating that the conversion of Dr-Nb(III) to Dr-Nb(II)-NO is limited by the OH^--based catalysis. From the dependence of k_lim on [OH^-], the value of the second-order rate constant k_OH- was obtained (5.2 × 10³ M^-1 s^-1). Reductive nitrosylation of Dr-Nb(III) leads to the inactivation of two NO molecules: one being converted to HNO_2, and the other being tightly bound to the heme-Fe(II) atom.