Inorganic nitrate and nitrite from endogenous or dietary sources are metabolized in vivo to nitric oxide (NO) and other bioactive nitrogen oxides. The nitrate-nitrite-NO pathway is emerging as an important mediator of blood flow regulation, cell signaling, energetics and tissue responses to hypoxia. The latest advances in our understanding of the biochemistry, physiology and therapeutics of nitrate, nitrite and NO were discussed during a recent two-day meeting at the Nobel Forum, Karolinska Institutet in Stockholm.
a Determined by microwave spectroscopy. b Determined by electron diffraction. c Data obtained from the Cambridge Structural Database. d This value was constrained. e Data for second molecule. f Data for disordered (minor) component.
It is now well established that mammalian heme proteins are reactive with various nitrogen oxide species and that these reactions may play significant roles in mammalian physiology. For example the ferrous heme protein myoglobin (Mb) has been shown to reduce nitrite (NO2−) to nitric oxide (NO) under hypoxic conditions. We demonstrate here that the distal pocket histidine residue (His64) of horse heart metMbIII (i.e., ferric MbIII) has marked effects on the mode of nitrite ion coordination to the iron center. X-ray crystal structures were determined for the mutant proteins metMbIII H64V (2.0 Å resolution) and its nitrite ion adduct metMbIII H64V-nitrite (1.95 Å resolution), and metMbIII H64V/V67R (1.9 Å resolution) and its nitrite ion adduct metMbIII H64V/V67R-nitrite (2.0 Å resolution). These are compared to the known structures of wild type hh metMbIII and its nitrite ion adduct hh metMbIII-nitrite, which binds NO2− via an O-atom in a trans-FeONO configuration. Unlike wt metMbIII, no axial H2O is evident in either of the metMbIII mutant structures. In the ferric H64V-nitrite structure, replacement of the distal His residue with Val alters the binding mode of nitrite from the nitrito (O-binding) form in the wild-type protein to a weakly bound nitro (N-binding) form. Reintroducing a H-bonding residue in the H64V/V67R double mutant restores the O-binding mode of nitrite. We have also examined the effects of these mutations on reactivities of the metMbIIIs with cysteine as a reducing agent and of the (ferrous) MbIIs with nitrite ion under anaerobic conditions. The MbIIs were generated by reduction of the MbIII precursors in a second order reaction with cysteine, the rate constants for this step following the order H64V/V67R > H64V >> wt. The rate constants for the oxidation of the MbIIs by nitrite (giving NO as the other product) follow the order wt > H64V/V67R >> H64V and suggest a significant role of the distal pocket H-bonding residue in nitrite reduction.
Several cobalt nitrosyl porphyrins of the form (T(p/m-X)PP)Co(NO) (p/m-X = p-OCH(3) (1), p-CH(3) (2), m-CH(3) (3), p-H (4), m-OCH(3) (5), p-OCF(3) (6), p-CF(3) (7), p-CN (8)) have been synthesized in 30-85% yields by reaction of the precursor cobalt porphyrin with nitric oxide. Compounds 1-7 were also prepared by reaction of the precursor cobalt porphyrin with nitrosonium tetrafluoroborate followed by reduction with cobaltocene. Compounds 1-8 have been characterized by elemental analysis, IR and (1)H NMR spectroscopy, mass spectrometry, and UV-vis spectrophotometry. They are diamagnetic and display nu(NO) bands in CH(2)Cl(2) between 1681 and 1695 cm(-)(1). The molecular structure of 1, determined by a single-crystal X-ray crystallographic analysis, reveals a Co-N-O angle of 119.6(4) degrees. Crystals of 1 are monoclinic, P2/c, with a = 15.052(1) Å, b = 9.390(1) Å, c = 16.274(2) Å, beta = 111.04(1) degrees, V = 2146.8(4) Å(3), Z = 2, T = 228(2) K, D(calcd) = 1.271 g cm(-)(3), and final R1 = 0.0599 (wR2 = 0.1567, GOF = 1.054) for 3330 "observed" reflections with I >/= 2sigma(I). Cyclic voltammetry studies in CH(2)Cl(2) reveal that compounds 1-7 undergo two reversible oxidations and two reversible reductions at low temperature. This is not the case for compound 8, which undergoes two reversible reductions but an irreversible oxidation due to adsorption of the oxidized product onto the electrode surface. Combined electrochemistry-infrared studies demonstrate that each of the compounds 1-7 undergoes a first oxidation at the porphyrin pi ring system and a first reduction at either the metal center or the nitrosyl axial ligand. The formulation for the singly oxidized products of compounds 1-7 as porphyrin pi-cation radicals was confirmed by the presence of bands in the 1289-1294 cm(-)(1) region (for compounds 1-5), which are diagnostic IR bands for generation of tetraarylporphyrin pi-cation radicals.
The nitrite anion is known to oxidize and degrade hemoglobin (Hb). Recent literature reports suggest a nitrite reductase activity for Hb, converting nitrite into nitric oxide. Surprisingly, no structural information about Hb-nitrite interactions has been reported. We have determined the crystal structure of the ferric Hb-nitrite complex at 1.80 A resolution. The nitrite ligand adopts the uncommon O-nitrito binding mode. In addition, the nitrito conformations in the alpha and beta subunits are different, reflecting subtle effects of the distal His in orienting the nitrite ligand in the O-nitrito binding mode.
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