Tetrahydrobiopterin redox cycling in nitric oxide synthase: evidence supports a through-heme electron delivery.

The nitric oxide synthases (NOS) catalyze a two-step oxidation of l-arginine (Arg) to generate NO. In the first step, O activation involves one electron being provided to the heme by an enzyme-bound 6R-tetrahydro-l-biopterin cofactor (H B), and the H B radical must be reduced back to H B in order for NOS to continue catalysis. Although an NADPH-derived electron is used to reduce the H B radical, how this occurs is unknown.

Internal Spin Trapping of Thiyl Radical during the Complexation and Reduction of Cobalamin with Glutathione and Dithiothrietol.

The activation of cobalamin requires the reduction of Cbl(III) to Cbl(II). The reduction by glutathione and dithiothreitol was followed using visible spectroscopy and electron paramagnetic resonance. In addition the oxidation of glutathione was monitored.

Influence of heme-thiolate in shaping the catalytic properties of a bacterial nitric-oxide synthase.

Nitric-oxide synthases (NOS) are heme-thiolate enzymes that generate nitric oxide (NO) from L-arginine. Mammalian and bacterial NOSs contain a conserved tryptophan (Trp) that hydrogen bonds with the heme-thiolate ligand. We mutated Trp(66) to His and Phe (W66H, W66F) in B.

A new paramagnetic intermediate formed during the reaction of nitrite with deoxyhemoglobin.

The reduction of nitrite by deoxygenated hemoglobin chains has been implicated in red cell-induced vasodilation, although the mechanism for this process has not been established. We have previously demonstrated that the reaction of nitrite with deoxyhemoglobin produces a hybrid intermediate with properties of Hb(II)NO(+) and Hb(III)NO that builds up during the reaction retaining potential NO bioactivity. To explain the unexpected stability of this intermediate, which prevents NO release from the Hb(III)NO component, we had implicated the transfer of an electron from the β-93 thiol to NO(+) producing ·SHb(II)NO.

The quaternary hemoglobin conformation regulates the formation of the nitrite-induced bioactive intermediate and the dissociation of nitric oxide from this intermediate.

Deoxyhemoglobin reduces nitrite to nitric oxide (NO). In order to study the effect of the hemoglobin quaternary conformation on the nitrite reaction, we compared T-state deoxyhemoglobin with R-state deoxyhemoglobin produced by reacting hemoglobin with carboxypeptidase-A prior to deoxygenation. The nitrite reaction with deoxyhemoglobin was followed by chemiluminescence, electron paramagnetic resonance and visible spectroscopy.

Enzymatic properties of the ferredoxin-dependent nitrite reductase from Chlamydomonas reinhardtii. Evidence for hydroxylamine as a late intermediate in ammonia production.

The ferredoxin-dependent nitrite reductase from the green alga Chlamydomonas reinhardtii has been cloned, expressed in Escherichia coli as a His-tagged recombinant protein, and purified to homogeneity. The spectra, kinetic properties and substrate-binding parameters of the C. reinhardtii enzyme are quite similar to those of the ferredoxin-dependent spinach chloroplast nitrite reductase.

The interaction of spinach nitrite reductase with ferredoxin: a site-directed mutation study.

A series of site-directed mutants of the ferredoxin-dependent spinach nitrite reductase has been characterized and several amino acids have been identified that appear to be involved in the interaction of the enzyme with ferredoxin. In a complementary study, binding constants to nitrite reductase and steady-state kinetic parameters of site-directed mutants of ferredoxin were determined in an attempt to identify ferredoxin amino acids involved in the interaction with nitrite reductase. The results have been interpreted in terms of an in-silico docking model for the 1:1 complex of ferredoxin with nitrite reductase.

Intermediates detected by visible spectroscopy during the reaction of nitrite with deoxyhemoglobin: the effect of nitrite concentration and diphosphoglycerate.

The reaction of nitrite with deoxyhemoglobin (deoxyHb) results in the reduction of nitrite to NO, which binds unreacted deoxyHb forming Fe(II)-nitrosylhemoglobin (Hb(II)NO). The tight binding of NO to deoxyHb is, however, inconsistent with reports implicating this reaction with hypoxic vasodilation. This dilemma is resolved by the demonstration that metastable intermediates are formed in the course of the reaction of nitrite with deoxyHb.

Nitrite infusion increases cerebral blood flow and decreases mean arterial blood pressure in rats: a role for red cell NO.

It has been proposed that the reduction of nitrite by red cells producing NO plays a role in the regulation of vascular tone. This hypothesis was investigated in rats by measuring the effect of nitrite infusion on mean arterial blood pressure (MAP), cerebral blood flow (CBF) and cerebrovascular resistance (CVR) in conjunction with the accumulation of red cell NO. The relative magnitude of the effects on MAP and CBF as well as the time dependent changes during nitrite infusion are used to distinguish between the effects on the peripheral circulation and the effects on the cerebral circulation undergoing cerebral autoregulation.

Nitric oxide redox reactions and red cell biology.

Three hypotheses explain a role for red blood cells (RBCs) in delivering NO to the vasculature: (a) "the SNOHb hypothesis" involves the uptake of NO by RBCs with NO transferred from the heme to the beta-93 thiol in the R quaternary conformation, followed by the release to membrane thiols in the T quaternary conformation; and (b and c) "the nitrite hypotheses" bypass the intrinsic difficulties of transporting the highly reactive NO, by reutilizing the nitrite formed when NO reacts with oxygen. Deoxyhemoglobin reduces this nitrite back to NO. The distinction between the two nitrite mechanisms depends on the importance of intermediate species formed during nitrite reduction.

S-nitrosohemoglobin: a mechanism for its formation in conjunction with nitrite reduction by deoxyhemoglobin.

The formation of S-nitrosohemoglobin (SNOHb) in red cells has been a major point of contention among researchers in this field. We have delineated a new mechanism for the formation of SNOHb coupled to nitrite reduction by deoxygenated hemoglobin chains at low oxygen pressures. The establishment of this mechanism required the development of a chemiluminescence assay utilizing Cu(II) and ascorbic acid to directly measure nitrosothiols without any interference from nitrite or heme-NO.

Cross-linking with O-raffinose lowers oxygen affinity and stabilizes haemoglobin in a non-cooperative T-state conformation.

O-R-polyHbA(0) is an intra- and intermolecularly O-raffinose cross-linked derivative of deoxygenated human haemoglobin developed as an oxygen therapeutic. When compared with its native protein (HbA(0)), O-R-polyHbA(0) was found to be locked in the T (tense) quaternary conformation with a lower oxygen affinity, a reduced Bohr effect (50% of HbA(0)) and no measurable cooperativity (h=1). The kinetics of oxygen and CO binding to the protein indicate lower 'on' rates and faster 'off' rates than HbA(0) and the absence of effects of inositol hexaphosphate (IHP) on the kinetics.

Redox reactions of hemoglobin.

Redox reactions of hemoglobin have gained importance because of the general interest of the role of oxidative stress in diseases and the possible role of red blood cells in oxidative stress. Although electron paramagnetic resonance (EPR) is extremely valuable in studying hemoglobin redox reactions it has not been adequately used. We have focused in this review on the important contributions of EPR to our understanding of hemoglobin redox reactions.

Hemoglobin redox reactions and oxidative stress.

The role of hemoglobin in transporting oxygen is dependent on the reversible binding of oxygen to Fe(II) hemoglobin with molecular oxygen released at reduced oxygen pressures. The partially oxygenated hemoglobin formed with the release of oxygen from hemoglobin is susceptible to redox reactions where the functional Fe(II) heme is oxidized to Fe(III) and the substrate is reduced. In this article, we review two important redox reactions of hemoglobin and discuss the ramifications of these reactions.

Active nitric oxide produced in the red cell under hypoxic conditions by deoxyhemoglobin-mediated nitrite reduction.

Recent studies have generated a great deal of interest in a possible role for red blood cells in the transport of nitric oxide (NO) to the microcirculation and the vascular effect of this nitric oxide in facilitating the flow of blood through the microcirculation. Many questions have, however, been raised regarding such a mechanism. We have instead identified a completely new mechanism to explain the role of red cells in the delivery of NO to the microcirculation.

Site-specific cross-linking of human and bovine hemoglobins differentially alters oxygen binding and redox side reactions producing rhombic heme and heme degradation.

Chemically modified human or bovine hemoglobins (Hb) have been developed as oxygen-carrying therapeutics and are currently under clinical evaluation. Oxidative processes, which are in many cases enhanced when modifications are introduced that lower the oxygen affinity, can limit the safety of these proteins. We have carried out a systematic evaluation of two modified human Hbs (O-R-polyHbA(0) and DBBF-Hb) and one bovine Hb (polyHbBv).