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

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.


Introduction
Globins are globular ferrous heme-proteins that have evolved across all domains of life to sense, bind, store, and transport diatomic gases, as well as to catalyze the synthesis and scavenging of reactive nitrogen and oxygen species [1][2][3]. They are composed of six or four α-helical segments, which are shaped around heme with a 3/3-or 2/2-fold. In 3/3 globins, the A, B, and E α-helices face the heme distal side and the F, G, and H α-helices are in front of the proximal side. In 2/2 globins, two antiparallel pairs of α-helices B/E and G/H encompass the heme [2][3][4][5][6].
The physiological role(s) of the evolutionary conserved 10-stranded β-barrel Nbs is still uncertain [10][11][12][13]. Ferric Arabidopsis thaliana Nb has been hypothesized to transport and to release NO at the infection site after wounding and pathogenic infections; then, NO may reduce O 2 to the superoxide radical, increasing pathogen burden [10]. Moreover, ferric Mycobacterium tuberculosis and Homo sapiens Nbs (Mt-Nb(III) and Hs-Nb(III), respectively) have been reported to selectively bind NO and to catalyze peroxynitrite scavenging, protecting both organisms from peroxynitrite-mediated nitration [13,21].
To shed light on the NO chemistry of Nbs, the reductive nitrosylation of Mt-Nb(III) and Hs-Nb(III) has been investigated and analyzed in parallel with that of prototypical all-α-helical heme-proteins. Interestingly, the values of the dissociation equilibrium constant for NO binding to Mt-Nb(III) and Hs-Nb(III) (i.e., K) and of the second-order rate constant of the OH − -mediated conversion of Mt-Nb(III)-NO to Mt-Nb(II)-NO and of Hs-Nb(III)-NO to Hs-Nb(II)-NO (i.e., k OH− ) are closely similar to those of all-α-helical heme-proteins. The much faster value of the second-order rate constant for Mt-Nb(III) and Hs-Nb(III) nitrosylation (i.e., NO k on ) is likely related to the much more open heme distal side. The present results highlight the inactivation of two NO molecules: one being converted to HNO 2 , and another being tightly bound to the heme-Fe(II) atom. In this respect, Hs-Nb may act as a NO/O 2 sensor, modulating the transcriptional activity of THAP4.
To shed light on the NO chemistry of Nbs, the reductive nitrosylation of Mt-Nb(III) and Hs-Nb(III) has been investigated and analyzed in parallel with that of prototypical all-α-helical hemeproteins. Interestingly, the values of the dissociation equilibrium constant for NO binding to Mt-Nb(III) and Hs-Nb(III) (i.e., K) and of the second-order rate constant of the OH − -mediated conversion of Mt-Nb(III)-NO to Mt-Nb(II)-NO and of Hs-Nb(III)-NO to Hs-Nb(II)-NO (i.e., kOH−) are closely similar to those of all-α-helical heme-proteins. The much faster value of the second-order rate constant for Mt-Nb(III) and Hs-Nb(III) nitrosylation (i.e., NO kon) is likely related to the much more open heme distal side. The present results highlight the inactivation of two NO molecules: one being converted to HNO2, and another being tightly bound to the heme-Fe(II) atom. In this respect, Hs-Nb may act as a NO/O2 sensor, modulating the transcriptional activity of THAP4.

Results
The reductive nitrosylation of ferric heme-proteins is an alkaline-driven process involving two NO molecules, one being converted to HNO2 and the other being trapped tightly to the ferrous heme-Fe atom (Scheme 1) [22][23][24][25][26][27][28][29].  Upon mixing, Mt-Nb(III) and Hs-Nb(III) with the NO solutions, the absorbance changes of Mt-Nb(III) and Hs-Nb(III) nitrosylation (i.e., Mt-Nb(III)-NO and Hs-Nb(III)-NO formation; Scheme 1A) are likely lost in the dead-time of the rapid-mixing stopped-flow apparatus (~2 ms), as expected over this NO concentration range (spanning between 2.5 × 10 −5 and 1.0 × 10 −3 M) on the basis of the reported NO k on values [13] (Table 1). As a matter of fact, immediately after mixing (i.e., within the first 10 ms), the absorbance spectra are already those corresponding to Mt-Nb(III)-NO and Hs-Nb(III)-NO. Then, the absorbance spectra of Mt-Nb(III)-NO and Hs-Nb(III)-NO gradually change to those of Mt-Nb(II)-NO and Hs-Nb(II)-NO. Accordingly, the static difference absorbance spectra of Mt-Nb(III) minus Mt-Nb(II)-NO and Hs-Nb(III) minus Hs-Nb(II)-NO, obtained by keeping the Mt-Nb(III) and Hs-Nb(III) solutions under NO at p = 760.0 mm Hg, differ from the difference absorbance spectra obtained after rapid-mixing the Mt-Nb(III) and Hs-Nb(III) solutions with the NO solutions, since in the last case they correspond to those of Mt-Nb(III)-NO minus Mt-Nb(II)-NO and Hs-Nb(III)-NO minus Hs-Nb(II)-NO ( Figures 1A and 2A, respectively). This confirms that the observed time courses ( Figures 1B and 2B, respectively) reflect the conversion of Mt-Nb(III)-NO to Mt-Nb(II)-NO and of Hs-Nb(III)-NO to Hs-Nb(II)-NO. Table 1. Values of NO k on , K, and k OH− for the reductive nitrosylation of ferric heme-proteins.  [27]. f T = 20.0 • C. From [27]. g pH 7.2 and 22.0 • C. From [26]. h T = 22.0 • C. From [26]. i pH 7.3 and T = 20.0 • C. From [24]. j Room temperature. From [24]. k pH 9.2 and T = 20.0 • C. From [30]. l T = 20.0 • C. From [30]. m pH 8.79 and room temperature. From [23]. n Room temperature. From [23]. o Kinetics of NO binding to human Hb(III), human Hp1-1:Hb(III), and human Hp2-2:Hb(III) are very fast and therefore they are lost in the mixing time [23,28]. p Room temperature. From [23]. q pH 8.2 and T = 20 • C. From [28]. r T = 20 • C. [28].

Heme-Protein
The time-courses of Mt-Nb(III) and Hs-Nb(III) reductive nitrosylation are monophasic for 94 ± 6% of their course ( Figures 1B and 2B, respectively), corresponding to Scheme 1B-D. The values of the pseudo-first-order rate constant for Mt-Nb(III) and Hs-Nb(III) reductive nitrosylation (i.e., k) do not increase linearly with the NO concentration but tend to level off ( Figures 1C and 2C, respectively), highlighting the occurrence of a fast pre-equilibrium process followed by a rate-limiting step. The analysis of data shown in Figures 1C and 2C according to Equation (3) allowed us to determine the values of the dissociation equilibrium constant for Mt-Nb(III) and Hs-Nb(III) nitrosylation (i.e., K), and of the first-order rate constant limiting the reductive nitrosylation process (i.e., k lim ). The values of K are pH-independent (ranging between 4.4 (±0.5) × 10 −5 and 5.7 (±0.6) × 10 −5 M) ( Table 1)    The values of k lim for the reductive nitrosylation of Mt-Nb(III) and Hs-Nb(III) increase linearly with pH ( Figures 1D and 2D, respectively). The analysis of data according to Equation (4) allowed us to determine the values of k OH− for the OH − -catalyzed conversion of Mt-Nb(II)-NO + to Mt-Nb(II) and Hs-Nb(II)-NO + to Hs-Nb(II), respectively, from the slope of the linear plots of k lim versus [OH − ] ( Table 1). The intercept of the straight lines with the y axis (i.e., k H2O ) is close to 0 s −1 , indicating that OH − catalyzes the reductive nitrosylation of Mt-Nb(III) and Hs-Nb(III) more efficiently than H 2 O.

Discussion
Nitrobindins constitute a new class of heme-proteins, characterized by a peculiar all-β-barrel structural arrangement [10][11][12][13]. Thus far, they have been found in M. tuberculosis, A. thaliana, and H. sapiens, where they are a C-terminal domain of the single-chain nuclear protein THAP4. Since the heme iron is stably in the Fe(III) form, their possible physiological role has been proposed to be NO sensing [10,11,13]; therefore, the characterization of the interaction of Mt-Nb(III) and Hs-Nb(III) with NO and of their pseudo-enzymatic role as NO scavengers is of the utmost importance. Of note, Mt-Nb(III) and Hs-Nb(III) react quickly with NO, with the values of the second-order rate constant NO k on being 1.8 × 10 6 M −1 s −1 and 1.1 × 10 6 M −1 s −1 , respectively (Table 1); then, the Fe(III)-NO complex undergoes a slow reductive process, bringing about the formation of HNO 2 and Fe(II)-NO through a series of pH-dependent reactions (Scheme 1).

Materials
Hs-Nb and Mt-Nb were cloned, expressed, and purified as described previously [13]. The Hs-Nb and Mt-Nb concentration was determined spectrophotometrically using the following extinction coefficients at λ max = 407 nm: 100 and 147 mM cm −1 , respectively [13].
Gaseous NO (from Linde Caracciolossigeno S.r.l., Roma, Italy) was purified by flowing through a glass column packed with NaOH pellets, and then by passage through a trapping solution containing 20 mL of 5.0 M NaOH to remove traces of impurities; the NO pressure was 760.0 mmHg [43]. The stock NO solution was prepared anaerobically by keeping the degassed 2.0 × 10 −3 M bis-tris propane buffer solution (pH 7.0) under NO in a closed vessel at P = 760.0 mm Hg (T = 20.0 • C). The solubility of NO in the aqueous buffered solution is 2.05 × 10 −3 M, at P = 760.0 mm Hg and T = 20.0 • C [44]. The concentration of NO in the solution was determined, under anaerobic conditions and in the absence of the gaseous phase, by titration of ferrous horse heart Mb monitored by visible absorption spectroscopy [45].
All the other chemicals were obtained from Merck KGaA (Darmstadt, Germany). All products were of analytical grade and used without purification unless stated.
All the experiments were carried out with the SFM-20/MOS-200 rapid-mixing stopped-flow apparatus (BioLogic Science Instruments, Claix, France); the dead-time of the stopped-flow apparatus was~2 ms and the observation chamber was 1 cm.
It is noteworthy that Scheme 1 indeed represents a redox enzymatic process, where NO scavenging is associated with the reduction of the heme-Fe(III) and nitrogen oxidation from NO and HNO 2 ; thus, NO is the substrate and HNO 2 is the product, with the whole chemical transformation being rate-limited by the OH − -dependent rate-limiting step k OH− .

Conclusions
The present results highlight the role of Mt-Nb(III) and Hs-Nb(III) as NO scavengers through the reductive nitrosylation reaction, which leads to the inactivation of two NO molecules. In spite of their much higher reactivity with NO to form the Mt-Nb(III)-NO and Hs-Nb(III)-NO species, respectively, the rate-limiting step for the conversion of Fe(II)-NO + to Fe(II) and HNO 2 is essentially similar to that of all-α-helical heme-proteins, resulting only slightly faster than what was observed for human Hb, Methanosarcina acetivorans Pgb, and Glycine max legHb (Table 1). This indicates that the structural determinants controlling these reactions are different. Indeed, while the very different NO reactivity reflects the diverse structural organization of the heme distal pocket of all-α-helical and all-β-barrel heme-proteins, the similar reactivity of OH − is controlled by still undetermined factors. Reflecting the NO/O 2 ratio, the reductive nitrosylation of Mt-Nb(III) and Hs-Nb(III) occurs at high NO concentration, whereas the oxidation of Mt-Nb(II)-NO and Hs-Nb(II)-NO takes place at high O 2 levels. In this context, Hs-Nb may act as a NO/O 2 sensor, modulating the transcriptional activity of THAP4.

Conflicts of Interest:
The authors declare no conflict of interest.