NO Scavenging through Reductive Nitrosylation of Ferric Mycobacterium tuberculosis and Homo sapiens Nitrobindins

Abstract

Ferric nitrobindins (Nbs) selectively bind NO and catalyze the conversion of peroxynitrite to nitrate. In this study, we show that NO scavenging occurs through the reductive nitrosylation of ferric Mycobacterium tuberculosis and Homo sapiens nitrobindins (Mt-Nb(III) and Hs-Nb(III), respectively). The conversion of Mt-Nb(III) and Hs-Nb(III) to Mt-Nb(II)-NO and Hs-Nb(II)-NO, respectively, is a monophasic process, suggesting that over the explored NO concentration range (between 2.5 × 10−5 and 1.0 × 10−3 M), NO binding is lost in the mixing time (i.e., NOkon ≥ 1.0 × 106 M−1 s−1). The pseudo-first-order rate constant for the reductive nitrosylation of Mt-Nb(III) and Hs-Nb(III) (i.e., k) is not linearly dependent on the NO concentration but tends to level off, with a rate-limiting step (i.e., klim) whose values increase linearly with [OH−]. This indicates that the conversion of Mt-Nb(III) and Hs-Nb(III) to Mt-Nb(II)-NO and Hs-Nb(II)-NO, respectively, is limited by the OH−-based catalysis. From the dependence of klim on [OH−], the values of the second-order rate constant kOH− for the reductive nitrosylation of Mt-Nb(III)-NO and Hs-Nb(III)-NO were obtained (4.9 (±0.5) × 103 M−1 s−1 and 6.9 (±0.8) × 103 M−1 s−1, respectively). This process leads to the inactivation of two NO molecules: one being converted to HNO2 and another being tightly bound to the ferrous heme-Fe(II) atom.

Strategies of Pathogens to Escape from NO-Based Host Defense

“…This supports the notion that Nb can be part of the pool of proteins required to scavenge RNS produced by the host during the immune response. In this framework, M. tuberculosis Nb may become a novel therapeutic target for the treatment of M. tuberculosis infections as reported also for mycobacterial trHbN [ 47 , 48 , 168 , 169 , 170 , 171 , 172 , 173 , 174 , 175 , 176 ].…”

Nitrite Reductase Activity of Ferrous Nitrobindins: A Comparative Study

“…Ferric and ferrous Nbs (Nb(III) and Nb(II), respectively) bind reversibly to NO with similar combination rate constants ((1.4 ± 0.4) × 10 6 M −1 s −1 and (1.5 ± 0.6) × 10 6 M −1 s −1 , respectively); the very different values of the dissociation rate constant ((4.6 ± 2.6) × 10 1 s −1 and (5.7 ± 2.5) × 10 −2 s −1 , respectively) are at the root of the different affinities ((3.5 ± 2.5) × 10 −5 M and (3.5 ± 2.5) × 10 −8 M, respectively) [ 19 , 20 , 21 , 22 ]. Of note, NO binding to the sixth coordination position of the heme–Fe-atom induces the cleavage or the severe weakening of the fifth proximal His-Fe(II) bond, at a neutral pH; this process occurs only at low pH or in the presence of allosteric effectors in Mb and Hb, respectively [ 23 , 24 ].…”

“…Of note, NO binding to the sixth coordination position of the heme–Fe-atom induces the cleavage or the severe weakening of the fifth proximal His-Fe(II) bond, at a neutral pH; this process occurs only at low pH or in the presence of allosteric effectors in Mb and Hb, respectively [ 23 , 24 ]. Moreover, at alkaline pHs, NO facilitates the reduction of the heme–Fe(III)-atom leading to the nitrosylation of the ferrous metal center (Nb(II)-NO) with a OH − -dependent reaction via the transient formation of ferric nitrosylated Nb (Nb(III)-NO); concomitantly, one equivalent of HNO 2 is produced [ 20 , 22 ]. Furthermore, Nbs catalyze the isomerization of peroxynitrite to NO 3 − and NO 2 − in the absence and presence of CO 2 [ 19 , 25 , 26 ].…”

Structural and (Pseudo-)Enzymatic Properties of Neuroglobin: Its Possible Role in Neuroprotection

“…The most striking differences between the 2/2 and the 3/3 globin folds are: ( i ) the drastically shortened or absent A-helix, ( ii ) the alteration of the C-E region, and ( iii ) the presence of a long polypeptide segment, which precedes the short α-helix F where the proximal HisF8 residue is located coordinating the heme-Fe atom [ 2 , 4 , 7 , 8 , 21 ]. Over the last two decades, all β-barrel non-canonical globins (i.e., nitrophorins and nitrobindins) mainly devoted to NO metabolism have been reported [ 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. Of note, human nitrobindin, corresponding to the C -terminal domain of the nuclear protein THAP4, has been hypothesized to act as a NO sensor modulating the transcriptional activity residing at the N -terminus [ 25 , 26 , 27 , 28 , 29 ].…”

“…Over the last two decades, all β-barrel non-canonical globins (i.e., nitrophorins and nitrobindins) mainly devoted to NO metabolism have been reported [ 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. Of note, human nitrobindin, corresponding to the C -terminal domain of the nuclear protein THAP4, has been hypothesized to act as a NO sensor modulating the transcriptional activity residing at the N -terminus [ 25 , 26 , 27 , 28 , 29 ].…”