Mycobacterial and Human Ferrous Nitrobindins: Spectroscopic and Reactivity Properties

Abstract

Structural and functional properties of ferrous Mycobacterium tuberculosis (Mt-Nb) and human (Hs-Nb) nitrobindins (Nbs) were investigated. At pH 7.0 and 25.0 °C, the unliganded Fe(II) species is penta-coordinated and unlike most other hemoproteins no pH-dependence of its coordination was detected over the pH range between 2.2 and 7.0. Further, despite a very open distal side of the heme pocket (as also indicated by the vanishingly small geminate recombination of CO for both Nbs), which exposes the heme pocket to the bulk solvent, their reactivity toward ligands, such as CO and NO, is significantly slower than in most hemoproteins, envisaging either a proximal barrier for ligand binding and/or crowding of H2O molecules in the distal side of the heme pocket which impairs ligand binding to the heme Fe-atom. On the other hand, liganded species display already at pH 7.0 and 25 °C a severe weakening (in the case of CO) and a cleavage (in the case of NO) of the proximal Fe-His bond, suggesting that the ligand-linked movement of the Fe(II) atom onto the heme plane brings about a marked lengthening of the proximal Fe-imidazole bond, eventually leading to its rupture. This structural evidence is accompanied by a marked enhancement of both ligands dissociation rate constants. As a whole, these data clearly indicate that structural–functional relationships in Nbs strongly differ from what observed in mammalian and truncated hemoproteins, suggesting that Nbs play a functional role clearly distinct from other eukaryotic and prokaryotic hemoproteins.

Nitrite Reductase Activity of Ferrous Nitrobindins: A Comparative Study

“…The absorbance spectra of Mt -Nb(II)-NO, At -Nb(II)-NO, Dr -Nb(II)-NO, and Hs -Nb(II)-NO, obtained by NO 2 − reduction, overlapped with those achieved by flowing gaseous NO in Mt -Nb(II), At -Nb(II), Dr -Nb(II), and Hs -Nb(II) solutions. Interestingly, the absorbance spectra of Mt -Nb(II)-NO, At -Nb(II)-NO, Dr -Nb(II)-NO, and Hs -Nb(II)-NO reflected the cleavage or the severe weakening of the proximal His-Fe(II) bond as evidenced from EPR spectroscopy [ 21 , 23 , 24 ].…”

“…A dramatic example is represented by tetrameric Hs -Hb(II) in the R-state, Pc -Mb(II) and Hs -Nb(II), which all displayed values of k on(NO2−) ~ 6.0 M −1 s −1 while values of the CO-binding rate constant greatly differed, spanning between 1.0 × 10 7 M −1 s −1 and 1.0 × 10 5 M −1 s −1 ( Figure 5 , panel B, and Table 1 ). Although multiple conformations of both the distal and the proximal side of the heme pocket affect the CO-binding rate constant [ 79 , 80 ], a likely structural explanation has been attributed to the activation free energy for the ligand-induced movement of the Fe(II) atom into the heme plane, which is fairly low for tetrameric Hs -Hb(II) in the R-state, while it seems very high for Hs -Nb(II) [ 23 , 79 ]. As a matter of fact, it has been convincingly shown that a major contribution to the reactivity of CO with hemoproteins is represented by the energy required for the movement of the heme’s Fe atom from its unliganded position (about 0.5 Ǻ out of the heme plane on the proximal side) to the heme co-planar position in the CO-liganded form [ 2 , 79 , 81 ]; the major contribution stems from the steric repulsion between the imidazole of the proximal histidine and the heme pyrroles, which depends on the relative position and differs among various hemoproteins [ 2 ].…”

“…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 ]. 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 ].…”

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 ].…”