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Contains fulltext : 209065.pdf (Publisher’s version ) (Open Access) Anaerobic ammonium oxidation (anammox) is a microbial process responsible for significant nitrogen loss from the oceans and other ecosystems. The redox reactions at the heart of anammox are catalyzed by large multiheme enzyme complexes that rely on small cytochrome c proteins for electron shuttling. Among the most highly abundant of these cytochromes is a unique heterodimeric complex composed of class I and class II c-type cytochrome called NaxLS, which has distinctive biochemical and spectroscopic properties. Here, we present the 1.7 Å resolution crystal structure of this complex from the anammox organism Kuenenia stuttgartiensis (KsNaxLS). The structure reveals that the heme irons in each subunit exhibit a rare His/Cys ligation, which, as we show by substitution, causes the observed unusual spectral properties. Unlike its individual subunits, the KsNaxLS complex binds nitric oxide (NO) only at the distal heme side, forming 6cNO adducts. This is likely due to steric immobilization of the proximal heme binding motifs upon complex formation, a finding that may be of functional relevance, since NO is an intermediate in the central anammox metabolism. Pulldown experiments with K. stuttgartiensis cell-free extract showed that the KsNaxLS complex binds specifically to one of the central anammox enzyme complexes, hydrazine synthase, which uses NO as one of its substrates. It is therefore possible that the KsNaxLS complex plays a role in binding the volatile NO to retain it in the cell for transfer to hydrazine synthase. Alternatively, we propose that KsNaxLS may shuttle electrons to this enzyme complex. 22 september 2019

The structure-function relationship is at the heart of biology, and major protein deformations are correlated to specific functions. For ferrous heme proteins, doming is associated with the respiratory function in hemoglobin and myoglobins. Cytochrome c (Cyt c) has evolved to become an important electron-transfer protein in humans. In its ferrous form, it undergoes ligand release and doming upon photoexcitation, but its ferric form does not release the distal ligand, while the return to the ground state has been attributed to thermal relaxation. Here, by combining femtosecond Fe K-alpha and K-beta X-ray emission spectroscopy (XES) with Fe K-edge X-ray absorption near-edge structure (XANES), we demonstrate that the photocycle of ferric Cyt c is entirely due to a cascade among excited spin states of the iron ion, causing the ferric heme to undergo doming, which we identify. We also argue that this pattern is common to a wide diversity of ferric heme proteins, raising the question of the biological relevance of doming in such proteins.

Myoglobin (Mb), generally taken as the molecular model of monomeric globular heme-proteins, is devoted: (i) to act as an intracellular oxygen reservoir, (ii) to transport oxygen from the sarcolemma to the mitochondria of vertebrate heart and red muscle cells, and (iii) to act as a scavenger of nitrogen and oxygen reactive species protecting mitochondrial respiration. Here, the first evidence of ·NO inhibition of ferric Mb- (Mb(III)) mediated detoxification of peroxynitrite is reported, at pH 7.2 and 20.0 °C. ·NO binds to Mb(III) with a simple equilibrium; the value of the second-order rate constant for Mb(III) nitrosylation (i.e., ·NOkon) is (6.8 ± 0.7) × 104 M-1 s-1 and the value of the first-order rate constant for Mb(III)-NO denitrosylation (i.e., ·NOkoff) is 3.1 ± 0.3 s-1. The calculated value of the dissociation equilibrium constant for Mb(III)-NO complex formation (i.e., ·NOkoff/·NOkon = (4.6 ± 0.7) × 10-5 M) is virtually the same as that directly measured (i.e., ·NOK = (3.8 ± 0.5) × 10-5 M). In the absence of ·NO, Mb(III) catalyzes the conversion of peroxynitrite to NO3-, the value of the second-order rate constant (i.e., Pkon) being (1.9 ± 0.2) × 104 M-1 s-1. However, in the presence of ·NO, Mb(III)-mediated detoxification of peroxynitrite is only partially inhibited, underlying the possibility that also Mb(III)-NO is able to catalyze the peroxynitrite isomerization, though with a reduced rate (Pkon* = (2.8 ± 0.3) × 103 M-1 s-1). These data expand the multiple roles of ·NO in modulating heme-protein actions, envisaging a delicate balancing between peroxynitrite and ·NO, which is modulated through the relative amount of Mb(III) and Mb(III)-NO.

Nitric oxide (NO) is a reactive gas well-known as an air pollutant causing severe environmental problems. NO is also an important signaling molecule having a strong affinity towards heme proteins in the body. Taking this specialty as a model, a biomimetic membrane was developed by modification of the membrane surface with iron-porphyrin which depicts very similar structure to heme proteins. In this study, PVDF membrane was coated with synthesized (4-carboxyphenyl)-10,15,20-triphenyl-porphyrin iron(III) chloride (FeCTPP) to promote NO fixation on the surface. The coated membrane was characterized in terms of ATR-IR spectra, contact angle measurement, chemical composition, and morphological structure. Contact angle of original PVDF first decreased sharply after plasma treatment and surface polymerization steps but after incorporation of FeCTPP, the surface acquired its hydrophobicity again. NO binding capability of modified membrane surface was evaluated on the basis of X-ray Photoelectron. Upon exposure to NO gas, a chemical shift of Fe+3 and appearance of new N peak was observed due to the electron transfer from NO ligand to Fe ion with the attachment of nitrosyl group to FeCTPP. This modification brings the functionality to the membrane for being used in biological systems such as membrane bioreactor material in biological NO removal technology. (C) 2017 Elsevier B.V. All rights reserved.

Under oxygen-limiting conditions, the marine bacterium Dinoroseobacter shibae DFL12T generates energy via denitrification, a respiratory process in which nitric oxide (NO) is an intermediate. Accumulation of NO may cause cytotoxic effects. The response to this nitrosative (NO-triggered) stress is controlled by the Crp/Fnr-type transcriptional regulator DnrF. We analyzed the response to NO and the mechanism of NO sensing by the DnrF regulator. Using reporter gene fusions and transcriptomics, here we report that DnrF selectively repressed nitrate reductase (nap) genes, preventing further NO formation. In addition, DnrF induced the expression of the NO reductase genes (norCB), which promote NO consumption. We used UV-visible and EPR spectroscopy to characterize heme binding to DnrF and subsequent NO coordination. DnrF detects NO via its bound heme cofactor. We found that the dimeric DnrF bound one molecule of heme per subunit. Purified recombinant apo-DnrF bound its target promoter sequences (napD, nosR2, norC, hemA, and dnrE) in electromobility shift assays, and we identified a specific palindromic DNA-binding site 5′-TTGATN4ATCAA-3′ in these target sequences via mutagenesis studies. Most importantly, successive addition of heme as well as heme and NO to purified recombinant apo-DnrF protein increased affinity of the holo-DnrF for its specific binding motif in the napD promoter. On the basis of these results, we propose a model for the DnrF-mediated NO stress response of this marine bacterium.

Binding of nitric oxide (NO) to metalloporphyrins and heme groups is important in biochemistry while challenging to describe accurately by density functional theory (DFT) calculations. Here, the structural and thermochemical aspect of NO binding to Co(II) and Mn(II) porphyrins is investigated by DFT and DFT-D (dispersion-corrected) calculations, supported by reliable coupled-cluster methodology (CCSD(T)), and critically correlated with the experimental data. It is argued that whereas the bonding of NO to Co(II) porphyrin is a simple radical recombination, the bonding of NO to Mn(II) porphyrin is accompanied by a crossing of spin states. For this reason, the spin-state conversion energy contributes to the Mn–NO bond energy, and the paradigmatic correlation between bond length and bond energy is violated for the considered nitrosyl complexes: the Mn–NO bond is (structurally) shorter by ∼0.2 Å, albeit (energetically) weaker by a few kcal/mol, compared with the Co–NO bond. Moreover, none of the many tested DFT methods can reproduce the Co–NO and Mn–NO bond energies simultaneously, except for calculations with B3LYP*-D3, TPSSh-D3, and M06-D3 methods supplemented with the proposed spin-state energy correction (to compensate for an error on the calculated spin-state conversion energy). The results of this study are important to appreciate the role of spin-state changes in ligand binding properties of heme-related models. They also highlight the need for accurate calculations for correct interpretation of experimental data, including the qualitative structure–energy relationship.

Background: Intraventricular haemorrhage (IVH) occurs in 60-70% of neonates weighing 500-750 g and 10-20% of those weighing 1,000-1,500 g. All forms of IVH have been associated with neurocognitive deficits. Both subarachnoid and intraventricular haemorrhages have been associated with delayed vasospasm leading to neurological deficits. Pathways linking haemoglobin release from blood clots to vasospasm include heme induced activation of inflammasomes releasing interleukin-1 (IL-1) which can cause calcium dependent and independent vasospasm. Free haemoglobin is a potent scavenger of nitric oxide. Depletion of nitric oxide, a potent endogenous vasodilator, has been associated with features of vasospasm. Hypothesis: In premature newborns intraventricular haemorrhage causes cerebral vasospasm and associated neurodisability via heme induced increased inflammasome mediated interleukin-1 production and nitric oxide depletion. Confirmation of hypothesis and implications: This hypothesis could be confirmed in the intraventricular haemorrhage animal model with visualisation of any associated vasospasm by angiography and in newborns with IVH by transcranial doppler ultrasonography and correlation with cerebrospinal fluid interleukin-1 and nitric oxide metabolite levels. Confirmation of the role of heme in activation of inflammasomes causing IL-1 production and nitric oxide binding could be achieved by measuring the effect of heme scavenging interventions on IL-1 levels and levels of nitric oxide metabolites. In addition to removal of the accumulated blood of an IVH by drainage, irrigation and fibrinolytic therapy intrathecal application of vasodilators and heme scavenging agents like haptoglobin and haemopexin and systemic treatment with inhibitors of inflammasomes like ruscogenin or telmisartan could be used to prevent and treat cerebral vasospasm and thus reduce the risk of associated brain injury in premature neonates.

Nitric oxide (NO) is a radical diatomic gas molecule that, at low concentrations, plays important signaling roles in both eukaryotes and bacteria. In recent years, it has become evident that bacteria respond to low levels of NO in order to modulate their group behavior. Many bacteria respond via NO ligation to a well-established NO sensor called H-NOX (heme-nitric oxide/oxygen binding domain). Many others, such as Pseudomonas aeruginosa, lack an annotated hnoX gene in their genome yet are able to respond to low levels of NO to disperse their biofilms. This suggests the existence of a previously uncharacterized NO sensor. In this study, we describe the discovery of a novel nitric oxide binding protein (NosP; NO-sensing protein), which is much more widely conserved in bacteria than H-NOX, as well as a novel NO-responsive pathway in P. aeruginosa. We demonstrate that biofilms of a P. aeruginosa mutant lacking components of the NosP pathway lose the ability to disperse in response to NO. Upon cloning, expressing, and purifying NosP, we find it binds heme and ligates to NO with a dissociation rate constant that is comparable to that of other well-established NO-sensing proteins. Moreover, we show that NO-bound NosP is able to regulate the phosphorelay activity of a hybrid histidine kinase that is involved in biofilm regulation in P. aeruginosa. Thus, here, we present evidence of a novel NO-responsive pathway that regulates biofilm in P. aeruginosa.

Iron-sulfur proteins are among the primary targets of nitric oxide in cells. Previous studies have shown that iron-sulfur clusters hosted by cysteine residues in proteins are readily disrupted by nitric oxide forming a protein-bound dinitrosyl iron complex, thiolate-bridged di-iron tetranitrosyl complex, or octanitrosyl cluster. Here we report that human mitochondrial protein Miner2 [2Fe-2S] clusters can bind nitric oxide without disruption of the clusters. Miner2 is a member of a new CDGSH iron-sulfur protein family that also includes two mitochondrial proteins: the type II diabetes-related mitoNEET and the Wolfram syndrome 2-linked Miner1. Miner2 contains two CDGSH motifs, and each CDGSH motif hosts a [2Fe-2S] cluster via three cysteine and one histidine residues. Binding of nitric oxide in the reduced Miner2 [2Fe-2S] clusters produces a major absorption peak at 422 nm without releasing iron or sulfide from the clusters. The EPR measurements and mass spectrometry analyses further reveal that nitric oxide binds to the reduced [2Fe-2S] clusters in Miner2, with each cluster binding one nitric oxide. Although the [2Fe-2S] cluster in purified human mitoNEET and Miner1 fails to bind nitric oxide, a single mutation of Asp-96 to Val in mitoNEET or Asp-123 to Val in Miner1 facilitates nitric oxide binding in the [2Fe-2S] cluster, indicating that a subtle change of protein structure may switch mitoNEET and Miner1 to bind nitric oxide. The results suggest that binding of nitric oxide in the CDGSH-type [2Fe-2S] clusters in mitochondrial protein Miner2 may represent a new nitric oxide signaling mode in cells.

Horse heart carboxymethylated-cyt[Formula: see text] (CM-cyt[Formula: see text] displays myoglobin-like properties due to the cleavage of the heme-Fe-Met80 axial bond. Here, reductive nitrosylation of CM-cyt[Formula: see text](III) between pH 8.5 and 9.5, at [Formula: see text] 20.0 C, is reported. Under anaerobic conditions, the addition of NO to CM-cyt[Formula: see text](III) leads to the transient formation of CM-cyt[Formula: see text](III)-NO in equilibrium with CM-cyt[Formula: see text](II)-NO[Formula: see text]. In turn, CM-cyt[Formula: see text](II)-NO[Formula: see text] is converted to CM-cyt[Formula: see text](II) by OH[Formula: see text]-based catalysis. Then, CM-cyt[Formula: see text](II) binds NO very rapidly leading to CM-cyt[Formula: see text](II)-NO. Kinetics of NO binding to CM-cyt[Formula: see text](III) is independent of the ligand concentration, [Formula: see text] values ranging between 3.6 ± 0.4 s[Formula: see text] and 7.1 ± 0.7 s[Formula: see text]. This indicates that the formation of the CM-cytc(III)-NO complex is rate-limited by the cleavage of the weak heme-Fe(III) distal bond (likely Lys79). The conversion of CM-cyt[Formula: see text](III)-NO to CM-cyt[Formula: see text](II)-NO is rate-limited by the OH[Formula: see text]-mediated reduction of CM-cyt[Formula: see text](II)-NO[Formula: see text] ([Formula: see text] (1.2 ± 0.1) × 103M[Formula: see text].s[Formula: see text]. Lastly, the very fast nitrosylation of CM-cyt[Formula: see text](II) takes place, values of [Formula: see text] ranging between[Formula: see text]5.3 × 106M[Formula: see text].s[Formula: see text] and 1.4 × 107M[Formula: see text].s[Formula: see text]. These results indicate that CM-cyt[Formula: see text] behaves as the cardiolipin-cyt[Formula: see text] complex highlighting the role of the sixth axial ligand of the heme-Fe atom in the modulation of the metal-based reactivity.

The ability of hemoproteins to discriminate between diatomic molecules, and the subsequent affinity for their chosen ligand, is fundamental to the existence of life. These processes are often controlled by precise structural arrangements in proteins, with heme pocket residues driving reactivity and specificity. One such protein is cytochrome c', which has the ability to bind nitric oxide (NO) and carbon monoxide (CO) on opposite faces of the heme, a property that is shared with soluble guanylate cycle. Like soluble guanylate cyclase, cytochrome c' also excludes O2 completely from the binding pocket. Previous studies have shown that the NO binding mechanism is regulated by a proximal arginine residue (R124) and a distal leucine residue (L16). Here, we have investigated the roles of these residues in maintaining the affinity for NO in the heme binding environment by using various time-resolved spectroscopy techniques that span the entire femtosecond-second temporal range in the UV-vis spectrum, and the femtosecond-nanosecond range by IR spectroscopy. Our findings indicate that the tightly regulated NO rebinding events following excitation in wild-type cytochrome c' are affected in the R124A variant. In the R124A variant, vibrational and electronic changes extend continuously across all time scales (from fs-s), in contrast to wild-type cytochrome c' and the L16A variant. Based on these findings, we propose a NO (re)binding mechanism for the R124A variant of cytochrome c' that is distinct from that in wild-type cytochrome c'. In the wider context, these findings emphasize the importance of heme pocket architecture in maintaining the reactivity of hemoproteins towards their chosen ligand, and demonstrate the power of spectroscopic probes spanning a wide temporal range.

Nitrate metabolism in Chlamydomonas reinhardtii involves THB1, a monomeric hemoglobin thought to function as a nitric oxide dioxygenase (NOD). NOD activity requires dioxygen and nitric oxide binding followed by a one-electron oxidation of the heme iron and nitrate release. Unlike pentacoordinate flavohemoglobins, which are efficient NODs, THB1 uses two iron axial ligands: the conserved proximal histidine and a distal lysine (Lys53). As a ligand in both the oxidized (ferric) and reduced (ferrous) states, Lys53 is expected to lower the reorganization energy associated with electron transfer and therefore facilitate reduction of the ferric enzyme. In ferrous THB1, however, Lys53 must be displaced for substrate binding. To characterize Lys53 dynamics, THB1 was studied at various pH, temperatures, and pressures by NMR spectroscopy. Structural information indicates that the protein fold and Lys53 environment are independent of the oxidation state. High-pressure NMR experiments provided evidence that displacement of Lys53 occurs through fast equilibrium (∼3-4 × 10

Nitric oxide binding and unbinding from myoglobin (Mb) is central to the function of the protein. By using reactive molecular dynamics (MD) simulations, the dynamics following NO dissociation were characterized in both time and space. Ligand rebinding can be described by two processes on the 10 ps and 100 ps timescale, which agrees with recent optical and X-ray absorption experiments. Explicitly including the iron out-of-plane (Fe-oop) coordinate is essential for a meaningful interpretation of the data. The proposed existence of an "Fe-oop/NO-bound" state is confirmed and assigned to NO at a distance of approximately 3 Å away from the iron atom. However, calculated XANES spectra suggest that it is diffcult to distinguish between NO close to the heme-Fe and positions further away in the primary site. Another elusive state, with Fe-ON coordination, was not observed experimentally because it is masked by the energetically more favorable but dissociative (4) A state in this region, which makes the Fe-ON local minimum unobservable in wild-type Mb. However, suitable active-site mutations may stabilize this state.

The nitric oxide (NO) binding properties of monolayers of four different protoporphyrins IX adsorbed on aluminum oxide surfaces have been investigated. XPS and AFM results are consistent with the presence of a monolayer of porphyrins, bound to the surface by their carboxylic acid groups and with the porphine ring oriented more or less perpendicular to the surface. Kelvin probe measurements were done in which the samples were exposed to varying NO concentrations. NO binds strongly to all porphyrins, but protoporphyrin IX zinc(II) (Zn-PP) shows the most promising response. The response rate could be increased by adding spacer molecules to the porphyrin layer. Low ppb levels of NO were easily detected in ambient conditions. The NO binding properties of Zn-PP compare favorably to those of other porphyrins which were previously used for NO sensing. This makes Zn-PP a new and promising candidate for this application. © 2012 Elsevier B.V. All rights reserved.

SUMMARY Aims and Methods: To determine the effect of aminosalicylic acid derivatives on the concentration of nitric oxide produced in a cell-free system, by the use of a sensitive and specific polarographic meter. Results: The aminosalicylic acid derivatives 3-ASA (IC50 100 μm), 4-ASA (IC50 350 μm) and 5-ASA (IC50 5 μm) all decreased the nitric oxide signal. These drugs had a similar inhibitory effect on the formation in vitro of nitrite from sodium nitroprusside (IC50 200 μm, 500 μm and 100 μm, respectively). Sulphasalazine (31.1 ± 5% decrease in signal at 1 mm) was less effective than 5-ASA, but sulphapyridine, N-acetyl 5-ASA, indomethacin and hydrocortisone produced no decrease in nitric oxide signal at all. Conclusions: Nitric oxide binding may be part of the mechanism by which ASA derivatives exert their therapeutic effect, and this work suggests that it may be an important factor in the pathogenesis of ulcerative colitis.

The chemical synthesis of the 184-residue ferric heme-binding protein nitrophorin 4 was accomplished by sequential couplings of five unprotected peptide segments using α-ketoacid-hydroxylamine (KAHA) ligation reactions. The fully assembled protein was folded to its native structure and coordinated to the ferric heme b cofactor. The synthetic holoprotein, despite four homoserine residues at the ligation sites, showed identical properties to the wild-type protein in nitric oxide binding and nitrite dismutase reactivity. This work establishes the KAHA ligation as a valuable and viable approach for the chemical synthesis of proteins up to 20 kDa and demonstrates that it is well-suited for the preparation of hydrophobic protein targets.

The heme cofactor in soluble guanylate cyclase (sGC) is a selective receptor for NO, an important signaling molecule in eukaryotes. The sGC heme domain has been localized to the N-terminal 194 amino acids of the beta1 subunit of sGC and is a member of a family of conserved hemoproteins, called the H-NOX family (Heme-Nitric Oxide and/or OXygen-binding domain). Three new members of this family have now been cloned and characterized, two proteins from Legionella pneumophila (L1 H-NOX and L2 H-NOX) and one from Nostoc punctiforme (Np H-NOX). Like sGC, L1 H-NOX forms a 5-coordinate Fe(II)-NO complex. However, both L2 H-NOX and Np H-NOX form temperature-dependent mixtures of 5- and 6-coordinate Fe(II)-NO complexes; at low temperature, they are primarily 6-coordinate, and at high temperature, the equilibrium is shifted toward a 5-coordinate geometry. This equilibrium is fully reversible with temperature in the absence of free NO. This process is analyzed in terms of a thermally labile proximal Fe(II)-His bond and suggests that in both the 5- and 6-coordinate Fe(II)-NO complexes of L2 H-NOX and Np H-NOX, NO is bound in the distal heme pocket of the H-NOX fold. NO dissociation kinetics for L1 H-NOX and L2 H-NOX have been determined and support a model in which NO dissociates from the distal side of the heme in both 5- and 6-coordinate complexes.

The influence of the heme iron coordination on nitric oxide binding dynamics was investigated for the myoglobin mutant H93G (H93G-Mb) by picosecond absorption and resonance Raman time-resolved spectroscopies. In the H93G-Mb, the glycine replacing the proximal histidine does not interact with the heme iron so that exogenous substituents like imidazole may coordinate to the iron at the proximal position. Nitrosylation of H93G-Mb leads to either 6- or 5-coordinate species depending on the imidazole concentration. At high concentrations, (imidazole)-(NO)-6-coordinate heme is formed, and the photoinduced rebinding kinetics reveal two exponential picosecond phases ( approximately 10 and approximately 100 ps) similar to those of wild type myoglobin. At low concentrations, imidazole is displaced by the trans effect leading to a (NO)-5-coordinate heme, becoming 4-coordinate immediately after photolysis as revealed from the transient Raman spectrum. In this case, NO rebinding kinetics remain bi-exponential with no change in time constant of the fast component whose amplitude increases with respect to the 6-coordinate species. Bi-exponential NO geminate rebinding in 5-coordinate H93G-Mb is in contrast with the single-exponential process reported for nitrosylated soluble guanylate cyclase (Negrerie, M., Bouzhir, L., Martin, J. L., and Liebl, U. (2001) J. Biol. Chem. 276, 46815-46821). Thus, our data show that the iron coordination state or the heme iron out-of-plane motion are not at the origin of the bi-exponential kinetics, which depends upon the protein structure, and that the 4-coordinate state favors the fast phase of NO geminate rebinding. Consequently, the heme coordination state together with the energy barriers provided by the protein structure control the dynamics and affinity for NO-binding enzymes.

Rhodobacter capsulatus cytochrome c' (RCCP) has been overexpressed in Escherichia coli, and its spectroscopic and ligand-binding properties have been investigated. It is concluded that the heterologously expressed protein is assembled correctly, as judged by UV-vis absorption, EPR, and resonance Raman (RR) spectroscopy of the unligated protein as well as forms in which the heme is ligated by CO or NO. To probe the oligomerization state of RCCP and its potential influence on heme reactivity, we have compared the properties of wild-type RCCP with a mutant (K42E) that lacks a salt bridge at the subunit interface. Analytical ultracentrifugation indicates that wild-type and K42E proteins are both monomeric in solution, contrary to the homodimeric structure of the crystalline state. Surprisingly, the K42E mutation produces a number of changes at the heme center (nearly 20 A distant), including perturbation of the ferric spin-state equilibrium and a change in the ferrous heme-nitrosyl complex from a six-coordinate/five-coordinate mixture to a predominantly five-coordinate heme-NO species. RR spectra indicate that ferrous K42E and wild-type RCCP both have relatively high Fe-His stretching frequencies, suggesting that the more favored five-coordinate heme-nitrosyl formation in K42E is not caused by a weaker Fe2+-His bond. Nevertheless, the altered reactivity of ferrous K42E with NO, together with its modified ferric spin state, shows that structural changes originating at the dimer interface can affect the properties of the heme center, raising the exciting possibility that intermolecular encounters at the protein surface might modulate the reactivity of cytochrome c' in vivo.

Bacterial hemoglobins and flavohemoglobins share a common globin fold but differ otherwise in structural and functional aspects. The bases of these differences were investigated through kinetic studies on oxygen, carbon monoxide, and nitric oxide binding. The novel bacterial hemoglobins from Clostridium perfringens and Campylobacter jejuni and the flavohemoglobins from Bacillus subtilis and Salmonella enterica serovar Typhi have been analyzed. Examination of the biochemical and ligand binding properties of these proteins shows a clear distinction between the two groups. Flavohemoglobins show a much greater tendency to autoxidation compared to bacterial hemoglobins. The differences in affinity for oxygen, carbon monoxide, and nitric oxide between bacterial hemoglobins and flavohemoglobins are mainly due to differences in the association rate constants. The second-order rate constants for oxygen and carbon monoxide binding to bacterial hemoglobins are severalfold higher than those for flavohemoglobins. A similar trend is observed for NO association with the oxidized iron(III) form of the proteins. No major differences are observed among the values obtained for the dissociation rate constants for the two groups of bacterial proteins studied, and these constants are all rather similar to those for myoglobin. Taken together, our data suggest that differences exist between the mechanisms of ligand binding to bacterial hemoglobins and flavohemoglobins, suggesting different functions in the cell.

We investigated the early events involved in loss of mitochondrial membrane potential (DeltaPsi(mt)) leading to apoptosis in cells derived from patients with acute lymphocytic leukemia after exposure to phenolic antioxidants. Using the nitric oxide binding dye diaminofluorescein-FM diacetate, we found that intracellular nitric oxide (NO) levels increased significantly within 4h after exposure to the antioxidants curcumin, carnosol, and quercetin. Inhibition of nitric oxide synthetase (NOS) activity with mercaptoethylguanidine increased the percentage of leukemia cells with depolarized mitochondria membranes after antioxidant treatment. These data suggest that NO production in the leukemia-derived cells may be a protective response to maintain DeltaPsi(mt) after antioxidant exposure and inhibition of NOS increases the disruption of mitochondrial homeostasis induced by the antioxidants.

This article has evaluated the published data regarding the efficacy of RBC transfusions in the critically ill. Taken together, these studies generally support conservative RBC transfusion strategies in critical care to reduce the risk of transfusion-related adverse effects. The TRICC trial has established the safety ofa restrictive transfusion strategy, suggesting that physicians could minimize exposure to allogeneic RBCs by lowering their transfusion threshold. Further research will add to the generalizability of this study and explore the possible mechanism to explain why RBC transfusions do not improve outcomes in the critically ill. Additional studies will be necessary to determine the effects of RBC storage time and the presence of allogeneic leukocytes in allogeneic RBC. The following conclusions are evident: 1. RBC transfusion does not improve tissue oxygen consumption consistently in critically ill patients, either globally or at the level of the micro-circulation. 2. RBC transfusion is not associated with improvements in clinical outcome in the critically ill and may result in worse outcomes in some patients. 3. Specific factors that identify patients who will improve from RBC transfusion are difficult to identify. 4. Lack of efficacy of RBC transfusion likely is related to storage time, increased endothelial adherence of stored RBCs, nitric oxide binding by free hemoglobin in stored blood, donor leukocytes, host inflammatory response, and reduced red cell deformability.

The nitrosylation of two water-soluble iron-porphyrins, the anionic Fe(III)-meso-tetrakis(p-sulfonatophenyl)porphyrin (FeTPPS(4)) and the cationic Fe(III)-meso-tetrakis(4-N-methylpyridiniumyl)porphyrin (FeTMPyP), by the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) was studied using optical absorption spectroscopy. The influence of ionic and non-ionic micelles on rates of nitric oxide transfer was investigated. Initially, the effect of the micelles on the pH-dependent equilibrium between monomeric and micro-oxo-dimeric species of the iron-porphyrins was examined. It is not affected in micelle-porphyrin systems with electric charges identical in sign. The non-ionic micelles of polidocanol induce a small negative pK shift. In contrast, the dimerization equilibrium of porphyrins in oppositely charged micellar phases is displaced to lower pH by approximately 2 units. Nitric oxide binding to monomers and micro-oxo-dimers was examined at pH 5.0 and 8.0, respectively. Contrary to nitrosylation by NO gas, SNAP induces reductive nitrosylation. There was no observed NO-Fe(III)porphyrin intermediate. Nitrosylation rates were obtained and compared in aqueous buffer and in micellar systems. Monomers nitrosylate much faster than micro-oxo-dimers. Oppositely charged micelles prevent nitrosylation of the iron-porphyrins or considerably enhance nitrosylation times. Nitrosylation rates are comparable to transnitrosylation rates between several S-nitrosothiols and thiol-containing proteins, suggesting biological relevance for the process.

Recombinant human serum albumin complexed with tetraphenylporphinatoiron(II) derivative, albumin-heme (rHSA-FeP), is a synthetic oxygen (O 2 )-carrying plasma hemoprotein, which becomes a new class of red blood cell substitute. The UV-vis. absorption and ESR spectroscopy revealed that rHSA-FeP formed six-coordinate nitrosyl complex after exposure of nitric oxide (NO) gas. Although the NO-binding affinity of rHSA-FeP (P 1/2 NO: 1.7 x 10 -6 Torr, pH 7.3, 25 °C) is 9-fold higher compared to that of hemoglobin (Hb), the administration of this artificial hemoprotein solution into anesthetized rat does not induce an acute increase in blood pressure (hypertension), which is often observed in Hb-based O 2 -carriers due to the depletion of NO (endothelial derived relaxing factor).

The formation of nitric oxide in biological systems has led to the discovery of a number of post-translational protein modifications that could regulate protein function or potentially be utilized as transducers of nitric oxide signaling. Principal among the nitric oxide-mediated protein modifications are: the nitric oxide-iron heme binding, the S-nitrosylation of reduced cysteine residues, and the C-nitration of tyrosine and tryptophan residues. With the exception of the nitric oxide binding to heme iron proteins, the other two modifications appear to require secondary reactions of nitric oxide and the formation of nitrogen oxides. The rapid development of analytical and immunological methodologies has allowed for the quantification of S-nitrosylated and C-nitrated proteins in vivo revealing an apparent selectivity and specificity of the proteins modified. This review is primarily focused upon the nitration of tyrosine residues discussing parameters that may govern the in vivo selectivity of protein nitration, and the potential biological significance and clinical relevance of this nitric oxide-mediated protein modification.

Nitric oxide (NO) has been used as a substrate analog to explore the structural and electronic determinants of enzymatic superoxide reduction at the mononuclear iron active site of Pyrococcus furiosus superoxide reductase (SOR) through the use of EPR, resonance Raman, Fourier transform IR, UV-visible absorption, and variable-temperature variable-field magnetic CD spectroscopies. The NO adduct of reduced SOR is shown to have a near-axial S = 3/2 ground state with E/D = 0.06 and D = 12 ± 2 cm −1 (where D and E are the axial and rhombic zero-field splitting parameters, respectively) and the UV-visible absorption and magnetic CD spectra are dominated by an out-of-plane NO − (π*)-to-Fe 3+ (dπ) charge–transfer transition, polarized along the zero-field splitting axis. Resonance Raman studies indicate that the NO adduct is six-coordinate with NO ligated in a bent conformation trans to the cysteinyl S , as evidenced by the identification of ν(N–O) at 1,721 cm −1 , ν(Fe–NO) at 475 cm −1 , and ν(Fe–S(Cys), at 291 cm −1 , via 34 S and 15 NO isotope shifts. The electronic and vibrational properties of the S = 3/2 {FeNO} 7 unit are rationalized in terms of a limiting formulation involving a high-spin ( S = 5/2) Fe 3+ center antiferromagnetically coupled to a ( S = 1) NO − anion, with a highly covalent Fe 3+ −NO − interaction. The results support a catalytic mechanism for SOR, with the first step involving oxidative addition of superoxide to form a ferric-peroxo intermediate, and indicate the important roles that the Fe spin state and the trans cysteinate ligand play in effecting superoxide reduction and peroxide release.

The reaction of iron(III) (meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (Fe(III)TMPyP) with nitric oxide (NO) was studied by electronic absorption spectroscopy, ESR, and electrochemical and spectroelectrochemical techniques in aqueous solutions with pH from 2.2 to 12.0. Fe(III)TMPyP has been found to undergo a reductive nitrosylation in all pHs, and the product of nitric oxide binding to the porphyrin has been determined as iron(II) porphyrin nitrosyl complex ([Fe(II)(NO)TMPyP]). The rate of the reductive nitrosylation exhibits a tendency to get faster with increase in pH. An intermediate species was observed around neutral pH by spectroelectrochemical technique and was proposed to be the iron(II) nitrosyl complex of the mu-oxo dimeric form of FeTMPyP, which is known to be a predominant in neutral solutions.

Neuroglobin is a recently discovered member of the globin superfamily. Combined electron paramagnetic resonance and optical measurements show that, in Escherichia colicell cultures with low O2 concentration overexpressing wild-type mouse recombinant neuroglobin, the heme protein is mainly in a hexacoordinated deoxy ferrous form (F8His-Fe2+-E7His), whereby for a small fraction of the protein the endogenous protein ligand is replaced by NO. Analogous studies for mutated neuroglobin (mutation of E7-His to Leu, Val, or Gln) reveal the predominant presence of the nitrosyl ferrous form. After sonication of the cells wild-type neuroglobin oxidizes rapidly to the hexacoordinated ferric form, whereas NO ligation initially protects the mutants from oxidation. Flash photolysis studies of wild-type neuroglobin and its E7 mutants show high recombination rates (k on) and low dissociation rates (k off) for NO, indicating a high intrinsic affinity for this ligand similar to that of other hemoglobins. Since the rate-limiting step in ligand combination with the deoxy-hexacoordinated wild-type form involves the dissociation of the protein ligand, NO binding is slower than for the related mutants. Structural and kinetic characteristics of neuroglobin and its mutants are analyzed. NO production in rapidly growing E. coli cell cultures is discussed.

New Zealand white rabbit cavernosal smooth muscle strips (n=6) were mounted in organ baths. Relaxations to nitric oxide (10(-7)-10(-4) mol/l) were measured and the same procedure was repeated on strips from rabbits 6 months after alloxan-induced diabetes (n=6). Transverse cavernosal sections were obtained from the same penises. Low and high resolution autoradiographs were prepared using [(3)H]-L-N(G)-nitroarginine (an index of nitric oxide binding sites) and analysed densitometrically. Histochemical analysis was performed on adjacent sections using NADPH diaphorase (an index of nitric oxide synthase activity). Nitric oxide relaxed control rabbit cavernosal smooth muscle strips in a concentration-dependent manner. Diabetic rabbit cavernosal smooth muscle strips were significantly (P

The binding of nitric oxide to ferric and ferrous Chromatium vinosum cytochrome c' was studied. The extinction coefficients for the ferric and ferrous nitric oxide complexes were measured. A binding model that included both a conformational change and dissociation of the dimer into subunits provided the best fit for the ferric cytochrome c' data. The NO (nitric oxide) binding affinity of the WT ferric form was found to be comparable to the affinities displayed by the ferric myoglobins and hemoglobins. Using an improved fitting model, positive cooperativity was found for the binding of NO to the WT ferric and ferrous forms, while anticooperativity was the case for the Y16F mutant. Structural explanations accounting for the binding are proposed. The NO affinity of ferrous cytochrome c' was found to be much lower than the affinities of myoglobins, hemoglobins, and pentacoordinate heme models. Structural factors accounting for the difference in affinities were analyzed. The NO affinity of ferrous cytochrome c' was found to be in the range typical of receptors and carriers. In addition, cytochrome c' was found to react with cytosolic light-irradiated membranes in the presence of succinate and carbon monoxide. With these results, a biochemical model of cytochrome c' functioning as a nitric oxide carrier was proposed.

Despite earlier work indicating otherwise, some recent reports have suggested that nitric oxide (NO) binds to hemoglobin cooperatively. In particular, it has been suggested that, under physiological conditions, NO binds to the high-affinity R-state hemoglobin as much as 100 times faster than to the low-affinity T-state hemoglobin. This rapid NO binding could provide a means of preserving NO bioactivity. However, using a flash-flow photolysis technique, we have determined that the rate of NO binding to normal adult R-state hemoglobin is (2.1 +/- 0.1) x 10(7) (s(-1) M(-1)), which is essentially the same as that reported for T-state NO binding. (c)2002 Elsevier Science (USA).

Gas phase studies offer an ideal medium whereby structural and reactivity properties of charged spe-cies may be unveiled in the absence of solvent, matrix or counterion effects. In this environment NO binds to iron(II)- and iron(III)-hemes with comparable kinetics and equilibrium parameters, conclu-sively elucidating the factors determining the widely different affinity in protic solvents or in heme proteins. IRMPD spectroscopy of the isolated species provides unambiguous characterization of the gaseous nitrosyl heme complexes.

Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a five- or six-coordinate Fe-NO complex. The H93G mutation eliminates the covalent attachment between the protein and the proximal ligand, allowing NO to bind H93G possibly from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side. The question of whether NO binds on the distal or proximal side was addressed by FTIR spectroscopy of the N-O vibrational frequency nuN(-O) for a set of Mb mutants that perturb the electrostatic environment of the heme pocket. Vibrational spectra of five- and six-coordinate MbNO complexes indicate that nu(N-O) shifts (by as much as 26 cm(-1)) to higher energies for the distal mutants H64V and H64V/H93G relative to the energies of wild-type and H93G MbNO, while nu(N-O) is not affected by the proximal side mutation S92A/H93G. This result suggests that NO binds on the distal side of heme in the five- and six-coordinate MbNO complexes of H93G. Additionally, values of the Fe-NO vibrational frequency nu(Fe-NO) as measured by resonance Raman spectroscopy are reported for the distal and proximal double mutants of H93G. These results suggest that nu(Fe-NO) is not very sensitive to mutations that perturb the electrostatic environment of the heme pocket, leading to the observation that nu(N-O) and nu(Fe-NO) are not quantitatively correlated for the MbNO complexes presented here. Furthermore, nu(N-O) and nu(Fe-NO) do not correlate well with equilibrium constants for imidazole binding to the five-coordinate MbNO complexes of the H93G double mutants. The data presented here do not appear to support the presence of pi-back-bonding or an inverse trans effect of NO binding in Mb mutants that alter the electrostatic environment of the heme pocket.

The resonance Raman (RR) spectra of nitrophorin 1 (NP1) from the saliva of the blood-sucking insect Rhodnius prolixus, in the absence and presence of nitric oxide (NO) and in the presence of cyanide (CN(-)), have been studied. The NP1 displayed RR spectra characteristic of six-coordinate high-spin (6cHS) ferric heme at room temperature and six-coordinate low-spin heme (6cLS) at low temperature (77 K). NO and CN(-) each bind to Fe(III), both ligands forming 6cLS complexes with NP1. The Fe(III)-NO stretching and bending vibrational frequencies of nitrosyl NP1 were identified at 591 and 578 cm(-1), respectively, on the basis of 15NO isotope shifts. These frequencies are typical of Fe-NO ferric heme proteins, indicating that the NP1 nitrosyl adduct has typical bond strength. Thus, the small NO release rate displayed by NP1 must be due to other protein interactions. Room and cryogenic temperature (77 K) RR spectroscopy and 13C, 15N, and 13C15N isotope substitutions have been used to determine vibrational mode frequencies associated with the Fe(III)-CN(-) bond for the cyano adducts at 454, 443, 397, and 357 cm(-1). The results were analyzed by normal mode calculations to support the assignment of the modes and to assess the NO and CN(-) binding geometries. The observed isotope shifts for the cyano NP1 are smaller than expected and reveal vibrational coupling of Fe(III)-CN(-) modes with heme modes. We also find that the observed frequencies are consistent with the presence of a nearly linear Fe(III)CN(-) linkage (173 degrees ) coexisting with a population with a bent structure (155 degrees ).

The nitrophorins comprise an unusual family of proteins that use ferric (Fe(III)) heme to transport highly reactive nitric oxide (NO) from the salivary gland of a blood sucking bug to the victim, resulting in vasodilation and reduced blood coagulation. We have determined structures of nitrophorin 4 in complexes with H2O, cyanide and nitric oxide. These structures reveal a remarkable feature: the nitrophorins have a broadly open distal pocket in the absence of NO, but upon NO binding, three or more water molecules are expelled and two loops fold into the distal pocket, resulting in the packing of hydrophobic groups around the NO molecule and increased distortion of the heme. In this way, the protein apparently forms a ‘hydrophobic trap’ for the NO molecule. The structures are very accurate, ranging between 1.6 and 1.4 A resolutions.

pH dependent nitric oxide binding of nitrophorin 1, an NO-carrier heme protein from Rhodnius prolixus has been studied by theoretical methods. p K a values of residues Asp30, Glu55, Asp70 and His59 were calculated to be 4.7, 5.2, 9.8 and 6.15, respectively using the Delphi package. On the basis of these results we concluded that the protonation of Asp30 and Glu55 is unlikely to have an effect on NO binding in the pH range of 5.0–8.3. Calculation of the electrostatic potential around the active site revealed that protonation of these residues results in a more positive potential at the distal side of the heme at pH 5.0 than that at pH 8.3 which destabilises the NO binding. The p K a value of Asp70 suggests this residue to be uncharged and therefore the water molecule can escape from hygrogen bonding (H-bonding) contacts with Asp70 and His59. p K a value of His59 was calculated to be 6.2 suggesting this residue to be protonated at pH 5.0. Protonation of His59 results in the breaking of His59–Fe bond. Conformational rearrangements at the proximal side were studied by the Monte Carlo conformational search. These calculations revealed that the water molecule can serve as the sixth ligand of the heme while the protonated His59 forms an H-bond with Asp70 and one of the heme propionates. Since the water molecule coordinated at pH 5.0 is a weak donor relative to the His59 ligated at pH 8.3 the binding of NO is stabilised at pH 5.0 which is in accordance with experimental data.

Here we show that in extrahepatic methionine adenosyltransferase replacement of a single amino acid (glycine 120) by cysteine is sufficient to create a functional nitric oxide binding site without affecting the kinetic properties of the enzyme. When wild-type and mutant methionine adenosyltransferase were incubated with S-nitrosoglutathione the activity of the wild-type remained unchanged whereas the activity of the mutant enzyme decreased markedly. The mutant enzyme was found to be S-nitrosylated upon incubation with the nitric oxide donor. Treatment of the S-nitrosylated mutant enzyme with glutathione removed most of the S-nitrosothiol groups and restored the activity to control values. In conclusion, our results suggest that functional S-nitrosylation sites can develop from existing structures without drastic or large-scale amino acid replacements.

Monte Carlo protein simulations with continuum solvation were used to explore the conformational mobility of NO within the active site of metmyoglobin. To the best of our knowledge this is the first application of a continuum solvation model for exploring protein binding sites. The usefulness of the Monte Carlo conformational analysis was demonstrated in comparative molecular dynamics simulations. Analysis of conformer populations revealed that Monte Carlo conformational analysis is more effective in mapping the relevant region of the potential surface. Distribution of low-energy conformers obtained for the metmyoglobin-NO complex was found to depend on the orientation of proximal His93. Different charge distributions corresponding to the two experimentally verified possible torsions of this proximal residue result in strong binding of NO or its release to a nearby hydrophobic trap. Conformer populations obtained by Monte Carlo conformational analysis were grouped into three main families: one, with the NO directly bound to the iron, appears when the CA-CB-CG-CD2 torsion of His93 is at its ligand binding value (-113 degrees); and two conformers exist where NO is trapped in a nearby hydrophobic pocket, the same cavity that was determined to be the geminate trap of CO in ferrous Mb as a result of the torsional flip of His93 to its ligand releasing state (-125 degrees). Based on this analysis, we suggest that the electrostatic rearrangement coupled to the conformational fluctuation of the proximal His leads to the geminate trapping of the ligand. Conformational rearrangement of the proximal side would provide the possibility of rebinding of the ligand to Fe.

The recombinant NO-binding heme protein, nitrophorin 1 (NP1) from the saliva of the blood-sucking insect, Rhodnius prolixus, has been studied by spectroelectrochemistry, EPR, NMR, and FTIR spectros...

Analysis of the iron K-edge X-ray absorption spectrum (XAS) of the “dark”, inactive form of nitrile hydratase (NHdk) from Rhodococcus sp. R312 confirms a 1:1 stoichiometry of nitric oxide bound to low-spin iron(III). We also report XAS analyses of four iron complexes of the pentadentate ligands 2,3,13,14-tetramethyl-4,8,12-triaza-3,12-pentadecadiene-2,14-dithiolate (L2-, also denoted as S2Me2N3(Pr,Pr)2-) and 2,12-dimethyl-3,7,11-triaza-2,11-tridecadiene-1,13-dithiolate (L‘2-, also denoted as S2N3(Pr,Pr)2-): five-coordinate FeIIL‘ and FeL+ and low-spin six-coordinate FeL(N3) and FeL(NO)+ (cationic species are PF6- salts). The XAS of FeL(N3) and FeL(NO)+ closely mimic the spectra of butyrate-stabilized active nitrile hydratase (NHlt) and NHdk, respectively. The 1s → 3d pre-edge peak is about twice as intense in five-coordinate FeL+ than for the remaining samples, suggesting that the iron in both NHlt and NHdk is six-coordinate. This peak and other edge features are 1 eV higher in energy for NHdk and FeL(NO...

When expressed in Escherichia coli, the heme domain [beta1(1-385)] of rat lung soluble guanylate cyclase (sGC) is isolated with a stoichiometric amount of bound heme [Zhao, Y., and Marletta, M. A. (1997) Biochemistry 36, 15959-15964]. Nitric oxide (NO) binding to the heme in beta1(1-385) leads to cleavage of the Fe-His bond and formation of a five-coordinate NO-heme complex. Addition of imidazole to the five-coordinate NO complex shifts the Soret peak from 399 to 420 nm, which appears to result from the formation of a six-coordinate NO complex. Removal of the added imidazole by gel filtration results in formation of the five-coordinate NO complex once again. The EPR spectrum of the putative six-coordinate NO complex has nine distinct derivative-shaped lines (a triplet of triplets), which is the signature spectrum of a six-coordinate NO complex with two nitrogen atoms as the axial ligands. [15N]Imidazole simplifies the six-coordinate NO complex EPR spectrum to six distinct derivative-shaped lines (a triplet of doublets), indicating that the other axial ligand in the six-coordinate NO complex is an imidazole molecule. These results show that NO binding to sGC not only leads to the cleavage of the Fe-His bond but also induces a conformational change which opens the heme proximal pocket large enough to accommodate an exogenous imidazole molecule. These observations have important implications for determining the NO activation mechanism of sGC.

A nitric oxide transport protein (nitrophorin I) from the salivary glands of the blood-sucking bug Rhodnius prolixus has been expressed as an insoluble form in Escherichia coli, reconstituted with heme, and characterized with respect to NO binding kinetics and equilibria. NO binding and absorption spectra for recombinant nitrophorin I were indistinguishable from those of the insect-derived protein. The degree of NO binding, the rate of NO release, and the Soret absorption maxima for nitrophorin I were all pH dependent. The NO dissociation constant rose 9-fold over the pH range 5.0-8.3, from 0.19 x 10(-6) to 1.71 x 10(-6). The NO dissociation rate rose 2500-fold between pH 5.0 and pH 8.3, from 1.2 x 10(-3) to 3.0 s(-1). Thus, the NO association rate must also be pH dependent and reduced at pH 5.0 by approximately 280-fold. These factors are consistent with nitrophorin function: NO storage in the apparent low pH of insect salivary glands and NO release into the tissue of the insect's host, where vasodilation is induced. The reversible nature of NO binding, which does not occur with most other heme proteins, and the apparent kinetic control of NO release are discussed. We also report crystals of nitrophorin I that are suitable for structure determination by X-ray crystallography. The most promising crystal form contains two protein molecules in the asymmetric unit and diffracts beyond 2.0 A resolution.

In this study, Tyr808 in GC-B (guanylate cyclase-B), a receptor of the CNP (C-type natriuretic peptide), has been shown to be a critical regulator of GC-B activity. In searching for phosphorylation sites that could account for suppression of GC-B activity by S1P (sphingosine-1-phosphate), mutations were introduced into several candidate serine/threonine and tyrosine residues. Although no novel phosphorylation sites that influenced the suppression of GC-B were identified, experiments revealed that mutations in Tyr808 markedly enhanced GC-B activity. CNP-stimulated activities of the Y808F and Y808A mutants were greater than 30-fold and 70-fold higher, respectively, than that of WT (wild-type) GC-B. The Y808E and Y808S mutants were constitutively active, expressing 270-fold higher activity without CNP stimulation than WT GC-B. Those mutations also influenced the sensitivity of GC-B to a variety of inhibitors, including S1P, Na3VO4 and PMA. Y808A, Y808E and Y808S mutations markedly weakened S1P- and Na3VO4-dependent suppression of GC-B activity, whereas Y808E and Y808S mutations rather elevated cGMP production. Tyr808 is conserved in all membrane-bound GCs and located in the niche domain showing sequence similarity to a partial fragment of the HNOBA (haem nitric oxide binding associated) domain, which is found in soluble GC and in bacterial haem-binding kinases. This finding provides new insight into the activation mechanism of GCs.

Neuronal nitric-oxide synthase (NOS-1) is a hemeprotein that generates NO and citrulline from L-arginine, O2, and NADPH. During catalysis, a majority of NOS-1 binds self-generated NO and converts to a ferrous-NO complex, which causes it to operate at a fraction of its maximum possible activity during the steady state (Abu-Soud, H. M., Wang, J., Rousseau, D. L., Fukuto, J., Ignarro, L. J., and Stuehr, D. J. (1995) J. Biol. Chem. 270, 22997-23006). To examine how NO complex formation affects the O2 response of NOS-1, we measured rates of NO synthesis and NADPH oxidation versus O2 concentration in the presence and absence of L-arginine. In the absence of L-arginine, NOS-1 catalyzed simple O2 reduction, and its heme iron displayed a typical affinity for O2 (estimated KmO2/= 40 microM, saturation at approximately 100 microM). In the presence of L-arginine, the rates of NO synthesis and NADPH oxidation were proportional to the O2 concentration over a much broader range (estimated KmO2 approximately 400 microM, saturation at approximately 800 microM), indicating that ferrous-NO complex formation altered the O2 response of NOS-1. Stopped-flow experiments revealed that the rate of ferrous-NO complex formation was relatively independent of the O2 concentration between 100 and 700 microM, while the rate of complex breakdown was directly proportional to O2 concentration. We conclude that the O2 sensitivity of the ferrous-NO complex governs the O2 response of NOS-1 and thus its activity during the steady state. This enables NOS-1 to couple its rate of NO synthesis to the O2 concentration throughout the physiologic range.