Crystallographic analysis of the interaction of nitric oxide with quaternary-T human hemoglobin
ABSTRACT In addition to interacting with hemoglobin as a heme ligand to form nitrosylhemoglobin, NO can react with cysteine sulfhydryl groups to form S-nitrosocysteine or cysteine oxides such as cysteinesulfenic acid. Both modes of interaction are very sensitive to the quaternary structure of hemoglobin. To directly view the interaction of NO with quaternary-T deoxyhemoglobin, crystallographic studies were carried out on crystals of deoxyhemoglobin that were exposed to gaseous NO under a variety of conditions. Consistent with previous spectroscopic studies in solution, these crystallographic studies show that the binding of NO to the heme groups of crystalline wild-type deoxyhemoglobin ruptures the Fe-proximal histidine bonds of the alpha-subunits but not the beta-subunits. This finding supports Perutz's theory that ligand binding induces tension in the alpha Fe-proximal histidine bond. To test Perutz's theory, deoxy crystals of the mutant hemoglobin betaW37E were exposed to NO. This experiment was carried out because previous studies have shown that this mutation greatly reduces the quaternary constraints that oppose the ligand-induced movement of the alpha-heme Fe atom into the plane of the porphyrin ring. As hypothesized, the Fe-proximal histidine bonds in both the beta- and the alpha-subunits remain intact in crystalline betaW37E after exposure to NO. With regard to S-nitrosocysteine or cysteine oxide formation, no evidence for the reaction of NO with any cysteine residues was detected under anaerobic conditions. However, when deoxyhemoglobin crystals are first exposed to air and then to NO, the appearance of additional electron density indicates that Cys93(F9)beta has been modified, most likely to cysteinesulfenic acid. This modification of Cys93(F9)beta disrupts the intrasubunit salt bridge between His146(HC3)beta and Asp94(FG1)beta, a key feature of the quaternary-T hemoglobin structure. Also presented is a reanalysis of our previous crystallographic studies [Chan, N.-L., et al. (1998) Biochemistry 37, 16459-16464] of the interaction of NO with liganded hemoglobin in the quaternary-R2 structure. These studies showed additional electron density at Cys93(F9)beta that was consistent with an NO adduct. However, for reasons discussed in this paper, we now believe that this adduct may be the Hb-S-N.-O-H radical intermediate and not Hb-S-N=O as previously suggested.
- [Show abstract] [Hide abstract]
ABSTRACT: Native horse heart cytochrome c (cytc) displays a very low reactivity toward ligands and does not exhibit catalytic properties. However, upon bovine cardiolipin (CL) binding, cytc achieves myoglobin-like properties. Here, NO binding to CL-cytc(III) between pH 7.2 and 9.5, at 20 °C, is reported. At pH 7.2, CL-cytc(III) undergoes reversible nitrosylation, whereas between pH 7.9 and 9.5 CL-cytc(III) undergoes irreversible reductive nitrosylation leading to the formation of CL-cytc(II)-NO. Over the whole pH range explored, first-order kinetics of NO binding to CL-cytc(III) (k = 9.3 s−1) indicates that ligand binding is limited by the cleavage of the weak heme-Fe distal bond. Between pH 7.9 and 9.5, nitrosylated CL-cytc(III) converts to the ligand-free ferrous derivative (CL-cytc(II)), this process being pH-dependent (hOH− = 3.0 × 103 M−1 s−1). Then, CL-cytc(II) converts to nitrosylated CL-cytc(II), in the presence of NO excess. The value of the second-order rate constant for CL-cytc(II) nitrosylation is essentially pH-independent, the average value of lon being 1.4 × 107 M−1 s−1. These results agree with the view that CL-cytc nitrosylation may play a role in apoptosis regulation. © 2014 IUBMB Life, 00(00):000–000, 2014International Union of Biochemistry and Molecular Biology Life 06/2014; DOI:10.1002/iub.1283 · 2.79 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Truncated hemoglobins (trHbs) are widely distributed in bacteria and plants and have been found in some unicellular eukaryotes. Phylogenetic analysis based on protein sequences shows that trHbs branch into three groups, designated N (or I), O (or II), and P (or III). Most trHbs are involved in the O2/NO chemistry and/or oxidation/reduction function, permitting the survival of the microorganism in the host. Here, a detailed comparative analysis of kinetics and/or thermodynamics of (i) ferrous Mycobacterium tubertulosis trHbs N and O (Mt-trHbN and Mt-trHbO, respectively), and Campylobacter jejuni trHb (Cj-trHbP) nitrosylation, (ii) nitrite-mediated nitrosylation of ferrous Mt-trHbN, Mt-trHbO, and Cj-trHbP, and (iii) NO-based reductive nitrosylation of ferric Mt-trHbN, Mt-trHbO, and Cj-trHbP is reported. Ferrous and ferric Mt-trHbN and Cj-trHbP display a very high reactivity towards NO; however, the conversion of nitrite to NO is facilitated primarily by ferrous Mt-trHbN. Values of kinetic and/or thermodynamic parameters reflect specific trHb structural features, such as the ligand diffusion pathways to/from the heme, the heme distal pocket structure and polarity, and the ligand stabilization mechanisms. In particular, the high reactivity of Mt-trHbN and Cj-trHbP reflects the great ligand accessibility to the heme center by two protein matrix tunnels and the E7-path, respectively, and the penta-coordination of the heme-Fe atom. In contrast, the heme-Fe atom of Mt-trHbO the ligand accessibility to the heme center of Mt-trHbO needs large conformational readjustments, thus limiting the heme-based reactivity. These results agree with different roles of Mt-trHbN, Mt-trHbO, and Cj-trHbP in vivo.PLoS ONE 07/2014; 9(7):e102811. DOI:10.1371/journal.pone.0102811 · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: This paper presents an algorithm that enables single-sensor operation of vector-controlled three-phase permanent-magnet-synchronous-motor voltage source inverter (VSI)-based drive systems. The closed-loop operation is performed through measuring only the VSI dc-link current. The measured VSI dc-link current is fed to a space-vector-based lookup table, which reconstructs the motor line currents. Three Luenberger-style observers are used to filter out the reconstructed motor line currents and estimate the rotor position and speed. The effect of observer bandwidth on the accuracy of the motor's estimated parameters is also investigated. The drive system's physical dynamic stiffness is used as a key metric to compare the drive system performance in single-sensor mode and fully sensored mode, in order to find the lowest steady-state speed limit for the single-sensor drive system operation.IEEE Transactions on Industry Applications 07/2014; 50(4):2602-2613. DOI:10.1109/TIA.2013.2296625 · 2.05 Impact Factor