Human nitrobindin: the first example of an all‐β‐barrel ferric heme‐protein that catalyzes peroxynitrite detoxification

Nitrobindins (Nbs), constituting a heme‐protein family spanning from bacteria to Homo sapiens, display an all‐β‐barrel structural organization. Human Nb has been described as a domain of the nuclear protein named THAP4, whose physiological function is still unknown. We report the first evidence of the heme‐Fe(III)‐based detoxification of peroxynitrite by the all‐β‐barrel C‐terminal Nb‐like domain of THAP4. Ferric human Nb (Nb(III)) catalyzes the conversion of peroxynitrite to NO3− and impairs the nitration of free l‐tyrosine. The rate of human Nb(III)‐mediated scavenging of peroxynitrite is similar to those of all‐α‐helical horse heart and sperm whale myoglobin and human hemoglobin, generally taken as the prototypes of all‐α‐helical heme‐proteins. The heme‐Fe(III) reactivity of all‐β‐barrel human Nb(III) and all‐α‐helical prototypical heme‐proteins possibly reflects the out‐to‐in‐plane transition of the heme‐Fe(III)‐atom preceding peroxynitrite binding. Human Nb(III) not only catalyzes the detoxification of peroxynitrite but also binds NO, possibly representing a target of reactive nitrogen species.

Nitrobindins (Nbs), constituting a heme-protein family spanning from bacteria to Homo sapiens, display an all-b-barrel structural organization. Human Nb has been described as a domain of the nuclear protein named THAP4, whose physiological function is still unknown. We report the first evidence of the heme-Fe(III)-based detoxification of peroxynitrite by the all-b-barrel C-terminal Nb-like domain of THAP4. Ferric human Nb (Nb (III)) catalyzes the conversion of peroxynitrite to NO À 3 and impairs the nitration of free L-tyrosine. The rate of human Nb(III)-mediated scavenging of peroxynitrite is similar to those of all-a-helical horse heart and sperm whale myoglobin and human hemoglobin, generally taken as the prototypes of all-a-helical heme-proteins. The heme-Fe(III) reactivity of all-b-barrel human Nb(III) and all-a-helical prototypical heme-proteins possibly reflects the out-to-in-plane transition of the heme-Fe(III)-atom preceding peroxynitrite binding. Human Nb(III) not only catalyzes the detoxification of peroxynitrite but also binds NO, possibly representing a target of reactive nitrogen species.
Here, the first evidence of the heme-Fe-based detoxification of peroxynitrite by the ferric all-b-barrel Cterminal Nb(III) domain of THAP4 (hereafter human Nb(III)) is reported. Human Nb(III) catalyzes efficiently the conversion of peroxynitrite to NO À 3 and impairs the peroxynitrite-mediated nitration of free Ltyrosine. These results point to a role of THAP4 in reactive nitrogen species chemistry.

Materials
The pReceiver-B03 vector containing the transcript variant 2 of H. sapiens Nb(III) domain (GeneCopoeia, Rockville, MD, USA) was used to amplify by PCR the Nb gene (fw_HindIII_NdeI: 5 0 -GCCCAAGCTTCATATGGAGCC CCCCAAG-3 0 and rv_BamHI: 5 0 -CGCGGATCCTTACGG GGTCAC-3 0 ). The fragment of 500 bp was first subcloned in the pBluescript KS(À) and finally cloned in the pET-28a (+) vector. The Escherichia coli BL21(DE3) strain was used to express the 6 9 His-tag-Nb in the presence of 0.2 mM d-aminolevulinic acid. The expression of the 6 9 His-tagged Nb was induced by adding 1 mM isopropyl-b-d-thiogalactoside for 16 h at 37°C. The bacterial pellet was lysed in 20 mM phosphate buffer pH 7.5, 140 mM NaCl, and 0.015% Tween-20, and the supernatant was loaded onto a His-Trap affinity chromatography column (GE Healthcare Bio-Sciences, Amersham, UK). The adsorbed 6 9 His-tag-Nb was eluted by a linear gradient of imidazole (20 mM phosphate buffer pH 7.4, 500 mM NaCl, and 10-1000 mM imidazole). The fractions containing the fusion protein were dialyzed against 20 mM phosphate buffer, pH 7.4, and analyzed by western blot using the primary anti-6 9 His-tag antibody (Thermo Fisher Scientific, Waltham, MA, USA). The human Nb(III) concentration was determined spectrophotometrically by the pyridine hemochromogen method [30]. Human apo-Nb was prepared by the acid-acetone method [30].

Methods
Peroxynitrite isomerization by human Nb(III) and apo-Nb was investigated by rapid mixing the human Nb(III) or apo-Nb solutions (final concentration ranging between 5.0 9 10 À6 and 3.5 9 10 À5 M) with the peroxynitrite solution (final concentration, 2.0 9 10 À4 M). Kinetics were recorded by using the SFM-20/MOS-200 rapid-mixing stopped-flow apparatus (BioLogic Science Instruments, Claix, France) monitoring absorbance changes at 302 nm [31]; the light path of the observation chamber was 10 mm, and the dead-time was 1.3 ms. In agreement with literature data [31,32], the absorbance at 302 nm decreased upon mixing the human Nb(III) and peroxynitrite solutions, reflecting the isomerization of peroxynitrite. No absorbance spectroscopic changes were observed in the Soret region in the course of the human Nb(III)-mediated isomerization of peroxynitrite.
Kinetic data were analyzed using the MATLAB program (The MathWorks Inc., Natick, MA, USA). The results are given as mean values of at least four experiments plus and minus the standard deviation.

Results and Discussion
Under all the experimental conditions, most of the time course of peroxynitrite isomerization (from 92% to 100%) was fitted to a single-exponential decay according to Eqn (1; Fig. 2, panel A). In fact, < 10% of the initial part of the time course of peroxynitrite isomerization was lost in the dead-time of the rapidmixing stopped-flow apparatus, depending on the decomposition rate.
The pseudo-first-order rate constant for human Nb (III)-mediated isomerization of peroxynitrite (i.e., k obs ) increases linearly with the protein concentration (Fig. 2, panel B). This suggests that (a) the formation of the transient human Nb(III)-OONO species represents the rate-limiting step in catalysis, and (b) the conversion of human Nb(III)-OONO to Nb(III) and NO À 3 and NO À 2 is faster than human Nb(III)-OONO formation by at least 10-fold. The analysis of the data shown in Fig. 2 (panel B), according to Eqn (2), allowed us to determine the values of the second-order rate constant for peroxynitrite isomerization by human Nb(III) (i.e., k on , corresponding to the slope of the linear plots) and of the first-order rate constant for the spontaneous peroxynitrite isomerization (i.e., k 0 , corresponding to the y-intercept of the linear plots) ( Table 1). Values of k 0 here determined agree with those previously reported [32,[36][37][38][39][40][41][42]45,46].
To confirm the role of the heme-Fe(III) atom in catalysis, values of k obs have been determined in the presence of human apo-Nb, which does not catalyze the peroxynitrite isomerization. Indeed, values of k obs obtained in the presence of human apo-Nb correspond to those of k 0 (Fig. 2, panel C) as reported, among others, for horse heart apo-Mb and human apo-Hb [36].
In the presence of human Nb(III), the values of the relative yield of NO À 3 and NO À 2 for the isomerization of peroxynitrite are 89 AE 2% and 12 AE 1%. However, in the absence of human Nb(III) and in the presence of human apo-Nb, the values of the relative yield of NO À 3 and NO À 2 are 68 AE 3% and 31 AE 2%, and 71 AE 2% and 28 AE 3%, respectively. These data well agree with those reported for peroxynitrite isomerization by ferric heme-proteins such as horse heart apo-Mb and human apo-Hb [36].
The pH dependence of k on and k 0 values for peroxynitrite isomerization allowed us to identify tentatively the species that preferentially react(s) with the heme-Fe(III) atom. Values of k on and k 0 increase upon decreasing pH (Fig. 3, panels A and B). The pK a values for the pH dependence of k on and of k 0 are 6.7 AE 0.2 and 6.8 AE 0.2, respectively (Fig. 3). The pK a values here determined agree well with those previously reported for the heme-protein-mediated isomerization of peroxynitrite [31,33,39,45,46].
The close similarity of the pH dependence of k on for the human Nb(III)-mediated isomerization of peroxynitrite (Fig. 3, panel A) and of k 0 for peroxynitrite isomerization in the absence of human Nb(III) (Fig. 3,  panel B) suggests that the HOONO species (Fig. 1 reacts preferentially with the heme-Fe(III) atom [31,33,38,46]. Since the absorbance spectrum of Nb (III) is unaffected by pH over the whole range explored (i.e., between pH 6.1 and 7.7; Fig. S1), it is unlikely that values of k on (Fig. 3, panel A) are affected by the acid-base equilibrium(a) of the ferric heme-protein.
To analyze the protective role of human Nb(III) against peroxynitrite-mediated nitration, the relative yield of nitro-L-tyrosine formed by the reaction of peroxynitrite with free L-tyrosine in the absence and presence of human Nb(III) and apo-Nb was determined. As expected, human Nb(III) protects dose-dependently free L-tyrosine against peroxynitrite-mediated nitration, whereas L-tyrosine nitration is not prevented by human apo-Nb (Fig. 4).
412 nm (human Nb(III)-NO). Moreover, human Nb (III)-NO can be converted to human Nb(III) by pumping off NO. In light of these considerations, data here reported highlight for the first time the capability of the Nb-like domain of human THAP4 protein to catalyze peroxynitrite scavenging, to impair the peroxynitritemediated nitration of free L-tyrosine, and to bind NO. Considering the structural organization of THAP4 [23,28,29], it can be speculated that THAP4 may play a role in the chemistry of reactive nitrogen species by coupling the heme-based Nb reactivity with the modulation of genes transcription. This somehow resembles other heme-proteins like NPAS2, in which the heme redox status controls NPAS heterodimerization with BMAL1 and, in turn, DNA binding and target gene expression [62,63]. Similar to A. thaliana Nb [22], human Nb is firmly in the ferric form possibly distinguishing among NO, CO, and O 2 . In fact, human Nb (III) selectively binds NO without recognizing CO and O 2 that are typical diatomic gaseous ligands of ferrous metal centers.
We are presently working on the functional analysis of the full-length human THAP4 protein as well as of its N-and/or C-terminal deleted forms to understand the molecular mechanisms underpinning THAP4 cellular functions.