Reactions of ferrous neuroglobin and cytoglobin with nitrite under anaerobic conditions

Recent evidence suggests that the reaction of nitrite with deoxygenated hemoglobin and myoglobin contributes to the generation of nitric oxide and S-nitrosothiols in vivo under conditions of low oxygen availability. We have investigated whether ferrous neuroglobin and cytoglobin, the two hexacoordinate globins from vertebrates expressed in brain and in a variety of tissues, respectively, also react with nitrite under anaerobic conditions. Using absorption spectroscopy, we find that ferrous neuroglobin and nitrite react with a second-order rate constant similar to that of myoglobin, whereas the ferrous heme of cytoglobin does not react with nitrite. Deconvolution of absorbance spectra shows that, in the course of the reaction of neuroglobin with nitrite, ferric Fe(III) heme is generated in excess of nitrosyl Fe(II)-NO heme as due to the low affinity of ferrous neuroglobin for nitric oxide. By using ferrous myoglobin as scavenger for nitric oxide, we find that nitric oxide dissociates from ferrous neuroglobin much faster than previously appreciated, consistently with the decay of the Fe(II)-NO product during the reaction. Both neuroglobin and cytoglobin are S-nitrosated when reacting with nitrite, with neuroglobin showing higher levels of S-nitrosation. The possible biological significance of the reaction between nitrite and neuroglobin in vivo under brain hypoxia is discussed.     

Substrate binding and the catalytic reactions in cbb3-type oxidases: The lipid membrane modulates ligand binding

Heme–copper oxidases (HCuOs) are the terminal components of the respiratory chain in the mitochondrial membrane or the cell membrane in many bacteria. These enzymes reduce oxygen to water and use the free energy from this reaction to maintain a proton-motive force across the membrane in which they are embedded. The heme–copper oxidases of the cbb₃-type are only found in bacteria, often pathogenic ones since they have a low K_m for O₂, enabling the bacteria to colonize semi-anoxic environments. Cbb₃-type (C) oxidases are highly divergent from the mitochondrial-like aa₃-type (A) oxidases, and within the heme–copper oxidase family, cbb₃ is the closest relative to the most divergent member, the bacterial nitric oxide reductase (NOR). Nitric oxide reductases reduce NO to N₂O without coupling the reaction to the generation of any electrochemical proton gradient. The significant structural differences between A- and C-type heme–copper oxidases are manifested in the lack in cbb₃ of most of the amino acids found to be important for proton pumping in the A-type, as well as in the different binding characteristics of ligands such as CO, O₂ and NO. Investigations of the reasons for these differences at a molecular level have provided insights into the mechanism of O₂ and NO reduction as well as the proton-pumping mechanism in all heme–copper oxidases. In this paper, we discuss results from these studies with the focus on the relationship between proton transfer and ligand binding and reduction. In addition, we present new data, which show that CO binding to one of the c-type hemes of CcoP is modulated by protein–lipid interactions in the membrane. These results show that the heme c-CO binding can be used as a probe of protein–membrane interactions in cbb₃ oxidases, and possible physiological consequences for this behavior are discussed.     

Nitrosylation of c heme in cd1-nitrite reductase is enhanced during catalysis

The reduction of nitrite into nitric oxide (NO) in denitrifying bacteria is catalyzed by nitrite reductase. In several species, this enzyme is a heme-containing protein with one c heme and one d₁ heme per monomer (cd₁NiR), encoded by the nirS gene.     

Nitrosylation Mechanisms of Mycobacterium tuberculosis and Campylobacter jejuni Truncated Hemoglobins N,O, and P

Truncated hemoglobins (trHbs) are widely distributed in bacteria and plants and have been found in some unicellular eukaryotes. Phylogenetic analysis based on protein sequences shows that trHbs branch into three groups, designated N (or I), O (or II), and P (or III). Most trHbs are involved in the O₂/NO chemistry and/or oxidation/reduction function, permitting the survival of the microorganism in the host. Here, a detailed comparative analysis of kinetics and/or thermodynamics of (i) ferrous Mycobacterium tubertulosis trHbs N and O (Mt-trHbN and Mt-trHbO, respectively), and Campylobacter jejuni trHb (Cj-trHbP) nitrosylation, (ii) nitrite-mediated nitrosylation of ferrous Mt-trHbN, Mt-trHbO, and Cj-trHbP, and (iii) NO-based reductive nitrosylation of ferric Mt-trHbN, Mt-trHbO, and Cj-trHbP is reported. Ferrous and ferric Mt-trHbN and Cj-trHbP display a very high reactivity towards NO; however, the conversion of nitrite to NO is facilitated primarily by ferrous Mt-trHbN. Values of kinetic and/or thermodynamic parameters reflect specific trHb structural features, such as the ligand diffusion pathways to/from the heme, the heme distal pocket structure and polarity, and the ligand stabilization mechanisms. In particular, the high reactivity of Mt-trHbN and Cj-trHbP reflects the great ligand accessibility to the heme center by two protein matrix tunnels and the E7-path, respectively, and the penta-coordination of the heme-Fe atom. In contrast, the heme-Fe atom of Mt-trHbO the ligand accessibility to the heme center of Mt-trHbO needs large conformational readjustments, thus limiting the heme-based reactivity. These results agree with different roles of Mt-trHbN, Mt-trHbO, and Cj-trHbP in vivo.     

Extreme differences between Hemoglobins I and II of the Clam Lucina pectinalis in their reactions with nitrite

The clam Lucina pectinalis supports its symbiotic bacteria by H₂S transport in the open and accessible heme pocket of Lucina Hb I and by O₂ transport in the narrow and crowded heme pocket of Lucina Hb II. Remarkably, air-equilibrated samples of Lucina Hb I were found to be more rapidly oxidized by nitrite than any previously studied Hb, while those of Lucina Hb II showed an unprecedented resistance to oxidation induced by nitrite. Nitrite-induced oxidation of Lucina Hb II was enabled only when O₂ was removed from its active site. Structural analysis revealed that O₂ “clams up” the active site by hydrogen bond formation to B10Tyr and other distal-side residues. Quaternary effects further restrict nitrite entry into the active site and stabilize the hydrogen-bonding network in oxygenated Lucina Hb II dimers. The dramatic differences in nitrite reactivities of the Lucina Hbs are not related to their O₂ affinities or anaerobic redox potentials, which were found to be similar, but are instead a result of differences in accessibility of nitrite to their active sites; i.e. these differences are due to a kinetic rather than thermodynamic effect. Comparative studies revealed heme accessibility to be a factor in human Hb oxidation by nitrite as well, as evidenced by variations of rates of nitrite-induced oxidation that do not correlate with R and T state differences and inhibition of oxidation rate in the presence of O₂. These results provide a dramatic illustration of how evolution of active sites with varied heme accessibility can moderate the rates of inner-sphere oxidative reactions of Hb and other heme proteins.     

Generating S-Nitrosothiols from Hemoglobin: MECHANISMS,CONFORMATIONAL DEPENDENCE,AND PHYSIOLOGICAL RELEVANCE*

In vitro, ferrous deoxy-hemes in hemoglobin (Hb) react with nitrite to generate nitric oxide (NO) through a nitrite reductase reaction. In vivo studies indicate Hb with nitrite can be a source of NO bioactivity. The nitrite reductase reaction does not appear to account fully for this activity because free NO is short lived especially within the red blood cell. Thus, the exporting of NO bioactivity both out of the RBC and over a large distance requires an additional mechanism. A nitrite anhydrase (NA) reaction in which N₂O₃, a potent S-nitrosating agent, is produced through the reaction of NO with ferric heme-bound nitrite has been proposed (Basu, S., Grubina, R., Huang, J., Conradie, J., Huang, Z., Jeffers, A., Jiang, A., He, X., Azarov, I., Seibert, R., Mehta, A., Patel, R., King, S. B., Hogg, N., Ghosh, A., Gladwin, M. T., and Kim-Shapiro, D. B. (2007) Nat. Chem. Biol. 3, 785–794) as a possible mechanism. Legitimate concerns, including physiological relevance and the nature of the mechanism, have been raised concerning the NA reaction. This study addresses these concerns demonstrating NO and nitrite with ferric hemes under near physiological conditions yield an intermediate having the properties of the purported NA heme-bound N₂O₃ intermediate. The results indicate that ferric heme sites, traditionally viewed as a source of potential toxicity, can be functionally significant, especially for partially oxygenated/partially met-R state Hb that arises from the NO dioxygenation reaction. In the presence of low levels of nitrite and either NO or a suitable reductant such as l-cysteine, these ferric heme sites can function as a generator for the formation of S-nitrosothiols such as S-nitrosoglutathione and, as such, should be considered as a source of RBC-derived and exportable bioactive NO.     

Structural characterization of a binuclear center of a Cu-containing NO reductase homologue from Roseobacter denitrificans: EPR and resonance Raman studies

Aerobic phototrophic bacterium Roseobacter denitrificans has a nitric oxide reductase (NOR) homologue with cytochrome c oxidase (CcO) activity. It is composed of two subunits that are homologous with NorC and NorB, and contains heme c, heme b, and copper in a 1:2:1 stoichiometry. This enzyme has virtually no NOR activity. Electron paramagnetic resonance (EPR) spectra of the air-oxidized enzyme showed signals of two low-spin hemes at 15 K. The high-spin heme species having relatively low signal intensity indicated that major part of heme b₃ is EPR-silent due to an antiferromagnetic coupling to an adjacent Cu_B forming a Fe-Cu binuclear center. Resonance Raman (RR) spectrum of the oxidized enzyme suggested that heme b₃ is six-coordinate high-spin species and the other hemes are six-coordinate low-spin species. The RR spectrum of the reduced enzyme showed that all the ferrous hemes are six-coordinate low-spin species. ν(Fe-CO) and ν(C-O) stretching modes were observed at 523 and 1969 cm⁻¹, respectively, for CO-bound enzyme. In spite of the similarity to NOR in the primary structure, the frequency of ν(Fe-CO) mode is close to those of aa₃- and bo₃-type oxidases rather than that of NOR.     

Blocking the K-pathway still allows rapid one-electron reduction of the binuclear center during the anaerobic reduction of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides

The K-pathway is one of the two proton-input channels required for function of cytochrome c oxidase. In the Rhodobacter sphaeroides cytochrome c oxidase, the K-channel starts at Glu101 in subunit II, which is at the surface of the protein exposed to the cytoplasm, and runs to Tyr288 at the heme a₃/Cu_B active site. Mutations of conserved, polar residues within the K-channel block or inhibit steady state oxidase activity. A large body of research has demonstrated that the K-channel is required to fully reduce the heme/Cu binuclear center, prior to the reaction with O₂, presumably by providing protons to stabilize the reduced metals (ferrous heme a₃ and cuprous Cu_B). However, there are conflicting reports which raise questions about whether blocking the K-channel blocks both electrons or only one electron from reaching the heme/Cu center. In the current work, the rate and extent of the anaerobic reduction of the heme/Cu center were monitored by optical and EPR spectroscopies, comparing the wild type and mutants that block the K-channel. The new data show that when the K-channel is blocked, one electron will still readily enter the binuclear center. The one-electron reduction of the resting oxidized (“O”) heme/Cu center of the K362M mutant, results in a partially reduced binuclear center in which the electron is distributed about evenly between heme a₃ and Cu_B in the R. sphaeroides oxidase. Complete reduction of the heme/Cu center requires the uptake of two protons which must be delivered through the K-channel.     

Myoglobin: a pseudo-enzymatic scavenger of nitric oxide

Myoglobin (Mb) is reported in biochemistry and physiology textbooks to act as an O₂ reservoir and to facilitate O₂ diffusion from capillaries to mitochondria, to sustain cellular respiration. Recently, it has been proposed that Mb is an intracellular scavenger of bioactive nitric oxide (NO), regulating its level in the skeletal and cardiac muscle and thereby protecting mitochondrial respiration, which is impaired by NO. This novel function of Mb is based on the rapid and irreversible reaction of ferrous oxygenated Mb (MbO₂) with NO yielding ferric oxidized Mb (metMb) and nitrate (NO₃^–). The efficiency of this process, which is postulated to depend on the superoxide (O₂^–) character acquired by O₂ once bound to the heme iron, may be enhanced by intramolecular diffusion of NO trapped momentarily into cavities of the protein matrix. O₂ can also react with ferrous nitrosylated Mb (MbNO), albeit very slowly, leading to metMb and NO₃^–. The O₂-dependent NO-detoxification process may be considered to be pseudo-enzymatic given that metMb obtained by the primary reaction of MbO₂ with NO is reduced back to ferrous Mb by a specific metMb-reductase, and can therefore repeat a cycle of NO conversion to harmless nitrate.     

Warfarin modulates the nitrite reductase activity of ferrous human serum heme–albumin

Human serum heme–albumin (HSA–heme–Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA–heme–Fe [HSA–heme–Fe(II)] is reported. The value of the second-order rate constant for the reduction of $ {\text{NO}}_{2}^{ - } $ to NO and the concomitant formation of nitrosylated HSA–heme–Fe(II) (i.e., k _on) is 1.3 M^?1 s^?1 at pH 7.4 and 20 °C. Values of k _on increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA–heme–Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA–heme–Fe(II) is (3.1 ± 0.4) × 10^?4 M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the $ {\text{NO}}_{2}^{ - } $ reductase activity of HSA–heme–Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(–heme–Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity.     

A Globin Domain in a Neuronal Transmembrane Receptor of Caenorhabditis elegans and Ascaris suum: MOLECULAR MODELING AND FUNCTIONAL PROPERTIES*

We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O₂ with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor.     

Bacterial denitrifying nitric oxide reductases and aerobic respiratory terminal oxidases use similar delivery pathways for their molecular substrates

The superfamily of heme?copper oxidoreductases (HCOs) include both NO and O₂ reductases. Nitric oxide reductases (NORs) are bacterial membrane enzymes that catalyze an intermediate step of denitrification by reducing nitric oxide (NO) to nitrous oxide (N₂O). They are structurally similar to heme?copper oxygen reductases (HCOs), which reduce O₂ to water. The experimentally observed apparent bimolecular rate constant of NO delivery to the deeply buried catalytic site of NORs was previously reported to approach the diffusion-controlled limit (10⁸–10⁹?M^?1?s^?1). Using the crystal structure of cytochrome-c dependent NOR (cNOR) from Pseudomonas aeruginosa, we employed several protocols of molecular dynamics (MD) simulation, which include flooding simulations of NO molecules, implicit ligand sampling and umbrella sampling simulations, to elucidate how NO in solution accesses the catalytic site of this cNOR. The results show that NO partitions into the membrane, enters the enzyme from the lipid bilayer and diffuses to the catalytic site via a hydrophobic tunnel that is resolved in the crystal structures. This is similar to what has been found for O₂ diffusion through the closely related O₂ reductases. The apparent second order rate constant approximated using the simulation data is ~5?×?10⁸?M^?1?s^?1, which is optimized by the dynamics of the amino acid side chains lining in the tunnel. It is concluded that both NO and O₂ reductases utilize well defined hydrophobic tunnels to assure that substrate diffusion to the buried catalytic sites is not rate limiting under physiological conditions.     

Parallel Pathways for Nitrite Reduction during Anaerobic Growth in Thermus thermophilus

Respiratory reduction of nitrate and nitrite is encoded in Thermus thermophilus by the respective transferable gene clusters. Nitrate is reduced by a heterotetrameric nitrate reductase (Nar) encoded along transporters and regulatory signal transduction systems within the nitrate respiration conjugative element (NCE). The nitrite respiration cluster (nic) encodes homologues of nitrite reductase (Nir) and nitric oxide reductase (Nor). The expression and role of the nirSJM genes in nitrite respiration were analyzed. The three genes are expressed from two promoters, one (nirSp) producing a tricistronic mRNA under aerobic and anaerobic conditions and the other (nirJp) producing a bicistronic mRNA only under conditions of anoxia plus a nitrogen oxide. As for its nitrite reductase homologues, NirS is expressed in the periplasm, has a covalently bound heme c, and conserves the heme d₁ binding pocket. NirJ is a cytoplasmic protein likely required for heme d₁ synthesis and NirS maturation. NirM is a soluble periplasmic homologue of cytochrome c₅₅₂. Mutants defective in nirS show normal anaerobic growth with nitrite and nitrate, supporting the existence of an alternative Nir in the cells. Gene knockout analysis of different candidate genes did not allow us to identify this alternative Nir protein but revealed the requirement for Nar in NirS-dependent and NirS-independent nitrite reduction. As the likely role for Nar in the process is in electron transport through its additional cytochrome c periplasmic subunit (NarC), we concluded all the Nir activity takes place in the periplasm by parallel pathways.     

Conversion of a heme-based oxygen sensor to a heme oxygenase by hydrogen sulfide: effects of mutations in the heme distal side of a heme-based oxygen sensor phosphodiesterase (Ec DOS)

The heme-based oxygen-sensor phosphodiesterase from Escherichia coli (Ec DOS), is composed of an N-terminal heme-bound oxygen sensing domain and a C-terminal catalytic domain. Oxygen (O₂) binding to the heme Fe(II) complex in Ec DOS substantially enhances catalysis. Addition of hydrogen sulfide (H₂S) to the heme Fe(III) complex in Ec DOS also remarkably stimulates catalysis in part due to the heme Fe(III)–SH and heme Fe(II)–O₂ complexes formed by H₂S. In this study, we examined the roles of the heme distal amino acids, M95 (the axial ligand of the heme Fe(II) complex) and R97 (the O₂ binding site in the heme Fe(II)–O₂ complex) of the isolated heme-binding domain of Ec DOS (Ec DOS-PAS) in the binding of H₂S under aerobic conditions. Interestingly, R97A and R97I mutant proteins formed an oxygen-incorporated modified heme, verdoheme, following addition of H₂S combined with H₂O₂ generated by the reactions. Time-dependent mass spectroscopic data corroborated the findings. In contrast, H₂S did not interact with the heme Fe(III) complex of M95H and R97E mutants. Thus, M95 and/or R97 on the heme distal side in Ec DOS-PAS significantly contribute to the interaction of H₂S with the Fe(III) heme complex and also to the modification of the heme Fe(III) complex with reactive oxygen species. Importantly, mutations of the O₂ binding site of the heme protein converted its function from oxygen sensor to that of a heme oxygenase. This study establishes the novel role of H₂S in modifying the heme iron complex to form verdoheme with the aid of reactive oxygen species.     

Crystal Structure of Dihydro-Heme d1 Dehydrogenase NirN from Pseudomonas aeruginosa Reveals Amino Acid Residues Essential for Catalysis

Many bacteria can switch from oxygen to nitrogen oxides, such as nitrate or nitrite, as terminal electron acceptors in their respiratory chain. This process is called “denitrification” and enables biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa, making it more resilient to antibiotics and highly adaptable to different habitats. The reduction of nitrite to nitric oxide is a crucial step during denitrification. It is catalyzed by the homodimeric cytochrome cd₁ nitrite reductase (NirS), which utilizes the unique isobacteriochlorin heme d₁ as its reaction center. Although the reaction mechanism of nitrite reduction is well understood, far less is known about the biosynthesis of heme d₁. The last step of its biosynthesis introduces a double bond in a propionate group of the tetrapyrrole to form an acrylate group. This conversion is catalyzed by the dehydrogenase NirN via a unique reaction mechanism. To get a more detailed insight into this reaction, the crystal structures of NirN with and without bound substrate have been determined. Similar to the homodimeric NirS, the monomeric NirN consists of an eight-bladed heme d₁-binding β-propeller and a cytochrome c domain, but their relative orientation differs with respect to NirS. His147 coordinates heme d₁ at the proximal side, whereas His323, which belongs to a flexible loop, binds at the distal position. Tyr461 and His417 are located next to the hydrogen atoms removed during dehydrogenation, suggesting an important role in catalysis. Activity assays with NirN variants revealed the essentiality of His147, His323 and Tyr461, but not of His417.     

Superoxide dismutase activity of macrocyclic polyamine complexes

Copper(II) and nickel(II) complexes of macrocyclic polyamine derivatives possessing partial oligopeptide-like structures are found to suppress the xanthine-xanthine oxidase-mediated reduction of nitroblue tetrazolium and also to suppress formazan formation by potassium superoxide. The activity in the superoxide dismutase assay is dependent on ring size, type and number of donor atoms, metal ion, and substituents on the macrocycles. Some of those are more active than the known O₂^? scavengers such as copper(II)-salicylate and copper(II)-amino acid (or peptide) complexes. Nickel (II)-naphthylmethyl-dioxo-[16]ane N₅, 13, 1 : 1 complex (NiH_?2L) is the most active among the 30 chelates examined.     

Cytochrome cd1 Nitrite Reductase NirS Is Involved in Anaerobic Magnetite Biomineralization in Magnetospirillum gryphiswaldense and Requires NirN for Proper d1 Heme Assembly

The alphaproteobacterium Magnetospirillum gryphiswaldense synthesizes magnetosomes, which are membrane-enveloped crystals of magnetite. Here we show that nitrite reduction is involved in redox control during anaerobic biomineralization of the mixed-valence iron oxide magnetite. The cytochrome cd₁-type nitrite reductase NirS shares conspicuous sequence similarity with NirN, which is also encoded within a larger nir cluster. Deletion of any one of these two nir genes resulted in impaired growth and smaller, fewer, and aberrantly shaped magnetite crystals during nitrate reduction. However, whereas nitrite reduction was completely abolished in the ΔnirS mutant, attenuated but significant nitrite reduction occurred in the ΔnirN mutant, indicating that only NirS is a nitrite reductase in M. gryphiswaldense. However, the ΔnirN mutant produced a different form of periplasmic d₁ heme that was not noncovalently bound to NirS, indicating that NirN is required for full reductase activity by maintaining a proper form of d₁ heme for holo-cytochrome cd₁ assembly. In conclusion, we assign for the first time a physiological function to NirN and demonstrate that effective nitrite reduction is required for biomineralization of wild-type crystals, probably by contributing to oxidation of ferrous iron under oxygen-limited conditions.     

The rate-limiting step in O2 reduction by cytochrome ba3 from Thermus thermophilus

Cytochrome ba₃ (ba₃) of Thermus thermophilus (T. thermophilus) is a member of the heme–copper oxidase family, which has a binuclear catalytic center comprised of a heme (heme a₃) and a copper (Cu_B). The heme–copper oxidases generally catalyze the four electron reduction of molecular oxygen in a sequence involving several intermediates. We have investigated the reaction of the fully reduced ba₃ with O₂ using stopped-flow techniques. Transient visible absorption spectra indicated that a fraction of the enzyme decayed to the oxidized state within the dead time (~ 1 ms) of the stopped-flow instrument, while the remaining amount was in a reduced state that decayed slowly (k = 400 s^? 1) to the oxidized state without accumulation of detectable intermediates. Furthermore, no accumulation of intermediate species at 1 ms was detected in time resolved resonance Raman measurements of the reaction. These findings suggest that O₂ binds rapidly to heme a₃ in one fraction of the enzyme and progresses to the oxidized state. In the other fraction of the enzyme, O₂ binds transiently to a trap, likely Cu_B, prior to its migration to heme a₃ for the oxidative reaction, highlighting the critical role of Cu_B in regulating the oxygen reaction kinetics in the oxidase superfamily. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Nitric oxide and peroxynitrite cause irreversible increases in the Km for oxygen of mitochondrial cytochrome oxidase: in vitro and in vivo studies

Mitochondrial cytochrome oxidase is competitively and reversibly inhibited by inhibitors that bind to ferrous heme, such as carbon monoxide and nitric oxide. In the case of nitric oxide, nanomolar levels inhibit cytochrome oxidase by competing with oxygen at the enzyme's heme-copper active site. This raises the K_m for cellular respiration into the physiological range. This effect is readily reversible and may be a physiological control mechanism. Here we show that a number of in vitro and in vivo conditions result in an irreversible increase in the oxygen K_m. These include: treatment of the purified enzyme with peroxynitrite or high (μM) levels of nitric oxide; treatment of the endothelial-derived cell line, b.End5, with NO; activation of astrocytes by cytokines; reperfusion injury in the gerbil brain. Studies of cell respiration that fail to vary the oxygen concentration systematically are therefore likely to significantly underestimate the degree of irreversible damage to cytochrome oxidase.