Regulation of nitrite reductase and lipid binding properties of cytoglobin by surface and distal histidine mutations.

Cytoglobin is a hemoprotein widely expressed in fibroblasts and related cell lineages with yet undefined physiological function. Cytoglobin, as other heme proteins, can reduce nitrite to nitric oxide (NO) providing a route to generate NO in vivo in low oxygen conditions. In addition, cytoglobin can also bind lipids such as oleic acid and cardiolipin with high affinity. These two processes are potentially relevant to cytoglobin function. Little is known about how specific amino acids contribute to nitrite reduction and lipid binding. Here we investigate the role of the distal histidine His81 (E7) and several surface residues on the regulation of nitrite reduction and lipid binding. We observe that the replacement of His81 (E7) greatly increases heme reactivity towards nitrite, with nitrite reduction rate constants of up to 1100 M-1s-1 for the His81Ala mutant. His81 (E7) mutation causes a small decrease in lipid binding affinity, however experiments on the presence of imidazole indicate that His81 (E7) does not compete with the lipid for the binding site. Mutations of the surface residues Arg84 and Lys116 largely impair lipid binding. Our results suggest that dissociation of His81 (E7) from the heme mediates the formation of a hydrophobic cavity in the proximal heme side that can accommodate the lipid, with important contributions of the hydrophobic patch around residues Thr91, Val105, and Leu108, whereas the positive charges from Arg84 and Lys116 stabilize the carboxyl group of the fatty acid. Gain and loss-of-function mutations described here can serve as tools to study in vivo the physiological role of these putative cytoglobin functions.

Direct measurement of nitric oxide (NO) production rates from enzymes using ozone-based gas-phase chemiluminescence (CL).

Nitric oxide (NO) chemiluminescence detectors (CLDs) are specialized and sensitive spectroscopic instruments capable of directly measuring NO flux rates. NO CLDs have been instrumental in the characterization of mammalian nitrite-dependent NO synthases. However, no detailed description of NO flux analysis using NO CLD is available. Herein, a detailed review of the NO CL methodology is provided with guidelines for measuring NO-production rates from aqueous samples, such as isolated enzymes or protein homogenates. Detailed description of the types of signals one can encounter, data processing, and potential pitfalls related to NO flux measurements will also be covered.

Carbonic anhydrase II does not regulate nitrite-dependent nitric oxide formation and vasodilation.

BACKGROUND AND PURPOSE: Although it has been reported that bovine carbonic anhydrase CAII is capable of generating NO from nitrite, the function and mechanism of CAII in nitrite-dependent NO formation and vascular responses remain controversial. We tested the hypothesis that CAII catalyses NO formation from nitrite and contributes to nitrite-dependent inhibition of platelet activation and vasodilation. EXPERIMENT APPROACH: The role of CAII in enzymatic NO generation was investigated by measuring NO formation from the reaction of isolated human and bovine CAII with nitrite using NO photolysis-chemiluminescence. A CAII-deficient mouse model was used to determine the role of CAII in red blood cell mediated nitrite reduction and vasodilation. KEY RESULTS: We found that the commercially available purified bovine CAII exhibited limited and non-enzymatic NO-generating reactivity in the presence of nitrite with or without addition of the CA inhibitor dorzolamide; the NO formation was eliminated with purification of the enzyme. There was no significant detectable NO production from the reaction of nitrite with recombinant human CAII. Using a CAII-deficient mouse model, there were no measurable changes in nitrite-dependent vasodilation in isolated aorta rings and in vivo in CAII-/- , CAII+/- , and wild-type mice. Moreover, deletion of the CAII gene in mice did not block nitrite reduction by red blood cells and the nitrite-NO-dependent inhibition of platelet activation. CONCLUSION AND IMPLICATIONS: These studies suggest that human, bovine and mouse CAII are not responsible for nitrite-dependent NO formation in red blood cells, aorta, or the systemic circulation.

The Zebrafish Cytochrome b5/Cytochrome b5 Reductase/NADH System Efficiently Reduces Cytoglobins 1 and 2: Conserved Activity of Cytochrome b5/Cytochrome b5 Reductases during Vertebrate Evolution.

Cytoglobin is a heme protein evolutionarily related to hemoglobin and myoglobin. Cytoglobin is expressed ubiquitously in mammalian tissues; however, its physiological functions are yet unclear. Phylogenetic analyses indicate that the cytoglobin gene is highly conserved in vertebrate clades, from fish to reptiles, amphibians, birds, and mammals. Most proposed roles for cytoglobin require the maintenance of a pool of reduced cytoglobin (FeII). We have shown previously that the human cytochrome b5/cytochrome b5 reductase system, considered a quintessential hemoglobin/myoglobin reductant, can reduce human and zebrafish cytoglobins ≤250-fold faster than human hemoglobin or myoglobin. It was unclear whether this reduction of zebrafish cytoglobins by mammalian proteins indicates a conserved pathway through vertebrate evolution. Here, we report the reduction of zebrafish cytoglobins 1 and 2 by the zebrafish cytochrome b5 reductase and the two zebrafish cytochrome b5 isoforms. In addition, the reducing system also supports reduction of Globin X, a conserved globin in fish and amphibians. Indeed, the zebrafish reducing system can maintain a fully reduced pool for both cytoglobins, and both cytochrome b5 isoforms can support this process. We determined the P50 for oxygen to be 0.5 Torr for cytoglobin 1 and 4.4 Torr for cytoglobin 2 at 25 °C. Thus, even at low oxygen tensions, the reduced cytoglobins may exist in a predominant oxygen-bound form. Under these conditions, the cytochrome b5/cytochrome b5 reductase system can support a conserved role for cytoglobins through evolution, providing electrons for redox signaling reactions such as nitric oxide dioxygenation, nitrite reduction, and phospholipid oxidation.

Molecular cloning, expression and biochemical characterization of periplasmic nitrate reductase from Campylobacter jejuni.

Campylobacter jejuni, a human gastrointestinal pathogen, uses nitrate for growth under microaerophilic conditions using periplasmic nitrate reductase (Nap). The catalytic subunit, NapA, contains two prosthetic groups, an iron sulfur cluster and a molybdenum cofactor. Here we describe the cloning, expression, purification, and Michaelis-Menten kinetics (kcat of 5.91 ± 0.18 s-1 and a KM (nitrate) of 3.40 ± 0.44 µM) in solution using methyl viologen as an electron donor. The data suggest that the high affinity of NapA for nitrate could support growth of C. jejuni on nitrate in the gastrointestinal tract. Site-directed mutagenesis was used and the codon for the molybdenum coordinating cysteine residue has been exchanged for serine. The resulting variant NapA is 4-fold less active than the native enzyme confirming the importance of this residue. The properties of the C. jejuni enzyme reported here represent the first isolation and characterization of an epsilonproteobacterial NapA. Therefore, the fundamental knowledge of Nap has been expanded.

Efficient Reduction of Vertebrate Cytoglobins by the Cytochrome b5/Cytochrome b5 Reductase/NADH System.

Cytoglobin is a heme-containing protein ubiquitous in mammalian tissues. Unlike the evolutionarily related proteins hemoglobin and myoglobin, cytoglobin shows a six-coordinated heme binding, with the heme iron coordinated by two histidine side chains. Cytoglobin is involved in cytoprotection pathways through yet undefined mechanisms, and it has recently been demonstrated that cytoglobin has redox signaling properties via nitric oxide (NO) and nitrite metabolism. The reduced, ferrous cytoglobin can bind oxygen and will react with NO in a dioxygenation reaction to form nitrate, which dampens NO signaling. When deoxygenated, cytoglobin can bind nitrite and reduce it to NO. This oxidoreductase activity could be catalytic if an effective reduction system exists to regenerate the reduced heme species. The nature of the physiological cytoglobin reducing system is unknown, although it has been proposed that ascorbate and cytochrome b5 could fulfill this role. Here we describe that physiological concentrations of cytochrome b5 and cytochrome b5 reductase can reduce human and fish cytoglobins at rates up to 250-fold higher than those reported for their known physiological substrates, hemoglobin and myoglobin, and up to 100-fold faster than 5 mM ascorbate. These data suggest that the cytochrome b5/cytochrome b5 reductase system is a viable reductant for cytoglobin in vivo, allowing for catalytic oxidoreductase activity.

Peroxidase activation of cytoglobin by anionic phospholipids: Mechanisms and consequences.

Cytoglobin (Cygb) is a hexa-coordinated hemoprotein with yet to be defined physiological functions. The iron coordination and spin state of the Cygb heme group are sensitive to oxidation of two cysteine residues (Cys38/Cys83) and/or the binding of free fatty acids. However, the roles of redox vs lipid regulators of Cygb's structural rearrangements in the context of the protein peroxidase competence are not known. Searching for physiologically relevant lipid regulators of Cygb, here we report that anionic phospholipids, particularly phosphatidylinositolphosphates, affect structural organization of the protein and modulate its iron state and peroxidase activity both conjointly and/or independently of cysteine oxidation. Thus, different anionic lipids can operate in cysteine-dependent and cysteine-independent ways as inducers of the peroxidase activity. We establish that Cygb's peroxidase activity can be utilized for the catalysis of peroxidation of anionic phospholipids (including phosphatidylinositolphosphates) yielding mono-oxygenated molecular species. Combined with the computational simulations we propose a bipartite lipid binding model that rationalizes the modes of interactions with phospholipids, the effects on structural re-arrangements and the peroxidase activity of the hemoprotein.

Exploring the mechanisms of the reductase activity of neuroglobin by site-directed mutagenesis of the heme distal pocket.

Neuroglobin (Ngb) is a six-coordinate globin that can catalyze the reduction of nitrite to nitric oxide. Although this reaction is common to heme proteins, the molecular interactions in the heme pocket that regulate this reaction are largely unknown. We have shown that the H64L Ngb mutation increases the rate of nitrite reduction by 2000-fold compared to that of wild-type Ngb [Tiso, M., et al. (2011) J. Biol. Chem. 286, 18277-18289]. Here we explore the effect of distal heme pocket mutations on nitrite reduction. For this purpose, we have generated mutations of Ngb residues Phe28(B10), His64(E7), and Val68(E11). Our results indicate a dichotomy in the reactivity of deoxy five- and six-coordinate globins toward nitrite. In hemoglobin and myoglobin, there is a correlation between faster rates and more negative potentials. However, in Ngb, reaction rates are apparently related to the distal pocket volume, and redox potential shows a poor relationship with the rate constants. This suggests a relationship between the nitrite reduction rate and heme accessibility in Ngb, particularly marked for His64(E7) mutants. In five-coordinate globins, His(E7) facilitates nitrite reduction, likely through proton donation. Conversely, in Ngb, the reduction mechanism does not rely on the delivery of a proton from the histidine side chain, as His64 mutants show the fastest reduction rates. In fact, the rate observed for H64A Ngb (1120 M(-1) s(-1)) is to the best of our knowledge the fastest reported for a heme nitrite reductase. These differences may be related to a differential stabilization of the iron-nitrite complexes in five- and six-coordinate globins.

Nitrite reductase and nitric-oxide synthase activity of the mitochondrial molybdopterin enzymes mARC1 and mARC2.

Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b5, and NADH-dependent cytochrome b5 reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar Kcat and Vmax values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production.