Article

Crystallographic Analysis of the Interaction of Nitric Oxide with Quaternary-T Human Hemoglobin

Department of Biochemistry, University of Iowa, Iowa City, Iowa, United States
Biochemistry (Impact Factor: 3.02). 02/2004; 43(1):118-32. DOI: 10.1021/bi030172j
Source: PubMed

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.

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    • "Electron paramagnetic resonance and photolysis-chemiluminescence techniques were used to quantify S-nitroso and nitrosylhemoglobin [30] [31]. Cristallographic analyses confirmed the binding of NO to the heme group of T state (deoxygenated) of hemoglobin while the NO binding to beta cysteine needs aerobic conditions [32]. "

    Preview · Article · Jul 2015
    • "The R-state to T-state transition for human HbNO was achieved upon addition of IHP, accompanied by a likely switch from six-coordination of heme Fe to five-coordination for about half of the hemes [12] [13]. Chan et al. prepared single crystals of T-state human HbNO and determined its structure [14]. They observed a cleavage of the proximal Fe–His bond in the a subunit (to yield five-coordinate heme nitrosyl) whereas this proximal Fe–His bond was retained in the b subunit. "
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    ABSTRACT: Human mesotrypsin is highly homologous to other mammalian trypsins, and yet is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. These findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    No preview · Article · Jul 2015 · Journal of Biological Chemistry
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    • "The R-state to T-state transition for human HbNO was achieved upon addition of IHP, accompanied by a likely switch from six-coordination of heme Fe to five-coordination for about half of the hemes [12] [13]. Chan et al. prepared single crystals of T-state human HbNO and determined its structure [14]. They observed a cleavage of the proximal Fe–His bond in the a subunit (to yield five-coordinate heme nitrosyl) whereas this proximal Fe–His bond was retained in the b subunit. "
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    ABSTRACT: Nitric oxide (NO) is a signaling agent that is biosynthesized in vivo. NO binds to the heme center in human hemoglobin (Hb) to form the HbNO adduct. This reaction of NO with Hb has been studied for many decades. Of continued interest has been the effect that the bound NO ligand has on the geometrical parameters of the resulting heme-NO active site. Although the crystal structure of a T-state human HbNO complex has been published previously, that of the high affinity R-state HbNO derivative has not been reported to date. We have crystallized and solved the three-dimensional X-ray structure of R-state human HbNO to 1.90 Å resolution. The differences in the FeNO bond parameters and H-bonding patterns between the α and β subunits contribute to understanding of the observed enhanced stability of the α(FeNO) moieties relative to the β(FeNO) moieties in human R-state HbNO.
    Full-text · Article · Apr 2014 · Nitric Oxide
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