Nitrite controls the release of nitric oxide in Pseudomonas aeruginosa cd1 nitrite reductase

Nitrite reductase (cd1NIR) from Pseudomonas aeruginosa, which catalyses the reduction of nitrite to nitric oxide (NO), contains a c-heme as the electron acceptor and a d1-heme where catalysis occurs. Reduction involves binding of nitrite to the reduced d1-heme, followed by dehydration to yield NO; release of NO and re-reduction of the enzyme close the cycle. Since NO is a powerful inhibitor of ferrous hemeproteins, enzymatic turnover demands the release of NO. We recently discovered that NO dissociation from the ferrous d1-heme is fast, showing that cd1NIR behaves differently from other hemeproteins. Here we demonstrate for the first time that the physiological substrate nitrite displaces NO from the ferrous enzyme, which enters a new catalytic cycle; this reaction depends on the conserved His369 whose role in substrate stabilization is crucial for catalysis. Thus we suggest that also in vivo the activity of cd1NIR is controlled by nitrite.     

Observation of fast release of NO from ferrous d₁ haem allows formulation of a unified reaction mechanism for cytochrome cd₁ nitrite reductases

Cytochrome cd1 nitrite reductase is a haem-containing enzyme responsible for the reduction of nitrite into NO, a key step in the anaerobic respiratory process of denitrification. The active site of cytochrome cd1 contains the unique d1 haem cofactor, from which NO must be released. In general, reduced haems bind NO tightly relative to oxidized haems. In the present paper, we present experimental evidence that the reduced d1 haem of cytochrome cd1 from Paracoccus pantotrophus releases NO rapidly (k=65-200 s(-1)); this result suggests that NO release is the rate-limiting step of the catalytic cycle (turnover number=72 s(-1)). We also demonstrate, using a complex of the d1 haem and apomyoglobin, that the rapid dissociation of NO is largely controlled by the d1 haem cofactor itself. We present a reaction mechanism proposed to be applicable to all cytochromes cd1 and conclude that the d1 haem has evolved to have low affinity for NO, as compared with other ferrous haems.     

Domain swing upon His to Ala mutation in nitrite reductase of Pseudomonas aeruginosa

The nitrite reductase (NIR) from Pseudomonas aeruginosa (NIR-Pa) is a soluble enzyme catalysing the reduction of nitrite (NO2(-)) to nitric oxide (NO). The enzyme is a 120 kDa homodimer, in which the monomers carry a c-heme domain and a d(1)-heme domain. The structures of the enzyme in both the oxidised and reduced state were solved previously and indicate His327 and His369 as putative catalytic residues. The kinetic characterisation of site-directed mutants has shown that the substitution of either one of these two His with Ala dramatically reduces the physiologically relevant reactivity towards nitrite, leaving the reactivity towards oxygen unaffected. The three-dimensional structures of P. aeruginosa NIR mutant H327A, and H369A in complex with NO have been solved by multiple wavelength anomalous dispersion (MAD), using the iron anomalous signal, and molecular replacement techniques. In both refined crystal structures the c-heme domain, whilst preserving its classical c-type cytochrome fold, has undergone a 60 degrees rigid-body rotation around an axis parallel with the pseudo 8-fold axis of the beta-propeller, and passing through residue Gln115. Even though the distance between the Fe ions of the c and d(1)-heme remains 21 A, the edge-to-edge distance between the two hemes has increased by 5 A. Furthermore the distal side of the d(1)-heme pocket appears to have undergone structural re-arrangement and Tyr10 has moved out of the active site. In the H369A-NO complex, the position and orientation of NO is significantly different from that of the NO bound to the reduced wild-type structure. Our results provide insight into the flexibility of the enzyme and the distinction between nitrite and oxidase reduction mechanisms. Moreover they demonstrate that the two histidine residues play a crucial role in the physiological activity of nitrite reduction, ligand binding and in the structural organisation of nitrite reductase from P. aeruginosa.     

Ancient hemes for ancient catalysts

Tetrapyrroles are essential molecules in living organisms and perform a multitude of functions in all kingdoms. Their synthesis is achieved in cells via a complex biosynthetic machinery which is unlikely to be maintained, if unnecessary. Here we propose that ancient hemes, such as the d₁-heme of cd₁ nitrite reductase or the siroheme of bacterial and plant nitrite and sulphite reductases, are molecular fossils which have survived the evolutionary pressure because their role is strategic for the organism where they are found today. The peculiar NO-releasing propensity of the d₁-heme of P. aeruginosa NIR, recently shown by our group is, in our opinion, an example of this strategy. The hypothesis is that the d₁-heme structure might be a pre-requisite for the fast rate of NO dissociation from the ferrous form, a property which is crucial to enzymatic activity and cannot be achieved with a more common b-type heme.Key words: d1-heme, porphyrin, siroheme, nitrite reductase, sulphite reductase, nitric oxide, evolutionPseudomonas aeruginosa is a Gram-negative bacterium commonly found in soil and water, well known for its metabolic versatility; under anaerobic conditions it can use nitrate and nitrite to produce energy via the denitrification pathway. In natural environments, denitrification is the part of the biological nitrogen cycle in which nitrate is transformed into nitrogen gas; reduction of nitrate occurs in four stages each catalyzed by a specific metalloenzyme.^1,2 P. aeruginosa is also an opportunistic pathogen, capable of causing serious infections in several hosts, such as humans and plants^3,4; pathogenesis, NO metabolism and denitrification are strictly related.^5,6The conversion of nitrite (NO₂^-) to nitric oxide (NO) is catalyzed in denitrifying bacteria by the periplasmic nitrite reductases (NIR).⁷ In P. aeruginosa NIR is a heme-containing enzyme (cd₁NIR) which produces NO in the active site where the unique d₁-heme cofactor (Fig. 1) is bound. This peculiar heme is synthesized from iron-protoporphyrin IX and belongs to the isobacteriochlorines subgroup;¹ it is exclusively found in this type of bacterial NIR.Open in a separate windowFigure 1Chemical structure of the d₁-heme.Reduction of nitrite involves binding of this molecule to the reduced d₁-heme, followed by dehydration to yield NO; release of NO and re-reduction of the enzyme close the cycle. An high affinity for nitrite (and anions) of the ferrous d₁-heme is a peculiar feature of cd₁NIR.⁷ However since the product NO is a powerful inhibitor of ferrous hemeproteins, enzymatic turnover demands the quick release of NO. In our recent paper⁸ we have shown that NO dissociates rapidly from the reduced form of the specialized d₁-heme of P. aeruginosa cd₁NIR. This unexpected result indicates that cd₁NIR behaves differently from other hemeproteins, since the rate of NO dissociation is by far faster (more than 100-fold) than that measured for any other heme in the ferrous state.^8–11Our hypothesis is that the d₁-heme structure might be a prerequisite for the fast rate of NO dissociation from the ferrous form, a property which cannot be achieved with a standard b-type heme.A major consequence of our finding is that this property of the d₁-heme is essential to avoid quasi-irreversible binding of NO to the reduced heme, which would jeopardize the physiological function of the enzyme evolved to scavenge nitrite, the toxic product of nitrate reduction. From the bioenergetic view-point, the main energy-generating step in denitrification is nitrate reduction (with a net H⁺ traslocation of 2H⁺/2e^-); thus, although a complex electron transfer chain is often present, the major biological role of the reductive steps downstream of nitrate reduction is likely to be nitrite scavenging.² If the complex of NO with reduced cd₁NIR was very long lived it would hamper further reaction cycles thus resulting in the accumulation of nitrite which is toxic for the bacterium. In line with this interpretation, we have also shown very recently¹² that nitrite is able to displace NO from the ferrous enzyme; thus substrate availability is the key factor that controls the enzyme turnover.From the standpoint of molecular evolution it is accepted that bacterial denitrification is an ancient metabolic pathway which existed even before oxygen became abundant in the athmosphere. Several reports pointed out that the enzymes involved in aerobic respiration derive from those involved in the denitrification pathway. Primitive denitrifying bacteria (similar to the extant Paracoccus denitrificans) can be considered as a common ancestral symbiotic prototype of the eukaryotic mitochondrion. Indeed there is compelling evidence that modern eukaryotic oxidases evolved from bacterial NO-reductase once oxygen became available as a major oxidant.^13,14In microrganisms, other “ancient” metabolisms are represented by sulphite and nitrite reduction pathways, which were well suited for a prebiotic photoreducing environment.¹⁵ Also in these pathways several enzymes are heme-containing proteins in which modified hemes, such as siroheme, are used as cofactors.¹⁶ Interestingly also in plants siroheme is a relevant porphyrin group,¹⁷ being the cofactor of plant nitrite and sulphite reductases, required for the assimilation of inorganic nitrogen and sulphur from the environment.Tetrapyrroles are essential molecules in living organisms and perform a multitude of functions in all kingdoms. Their biosynthesis is achieved in cells via branched pathways which are expensive in terms of energy consumption.^16–18 The single pathways are tightly regulated and often activated only “on demand” when the specific heme group is required. Therefore, parsimony suggests that a complex biosynthetic machinery is unlikely to be maintained, if unnecessary.We thus propose that these ancient hemes (such as the d₁-heme or the siroheme) are molecular fossils which have survived the evolutionary pressure because their role is strategic only for the organism where they are found today. The peculiar NO-releasing propensity of the d₁-heme of P. aeruginosa NIR shown by our group could be, in our opinion, an example of this strategy. A major challenge for the future is to unveil other uncommon features of these hemes.     

The catalytic mechanism of Pseudomonas aeruginosa cd1 nitrite reductase

The cd1 NiRs (nitrite reductases) are enzymes catalysing the reduction of nitrite to NO (nitric oxide) in the bacterial energy conversion denitrification process. These enzymes contain two distinct redox centres: one covalently bound c-haem, which is reduced by external electron donors, and another peculiar porphyrin, the d1-haem (3,8-dioxo-17-acrylate-porphyrindione), where nitrite is reduced to NO. In the present paper, we summarize the most recent results on the mechanism of nitrite reduction by the cd1 NiR from Pseudomonas aeruginosa. We discuss the essential catalytic features of this enzyme, with special attention to the allosteric regulation of the enzyme's activity and to the mechanism employed to avoid product inhibition, i.e. trapping of the active-site reduced haem by the product NO. These results shed light on the reactivity of cd1 NiRs and assign a central role to the unique d1-haem, present only in this class of enzymes.     

Pseudoazurin dramatically enhances the reaction profile of nitrite reduction by Paracoccus pantotrophus cytochrome cd1 and facilitates release of product nitric oxide

Cytochrome cd(1) is a respiratory nitrite reductase found in the periplasm of denitrifying bacteria. When fully reduced Paracoccus pantotrophus cytochrome cd(1) is mixed with nitrite in a stopped-flow apparatus in the absence of excess reductant, a kinetically stable complex of enzyme and product forms, assigned as a mixture of cFe(II) d(1)Fe(II)-NO(+) and cFe(III) d(1)Fe(II)-NO (cd(1)-X). However, in order for the enzyme to achieve steady-state turnover, product (NO) release must occur. In this work, we have investigated the effect of a physiological electron donor to cytochrome cd(1), the copper protein pseudoazurin, on the mechanism of nitrite reduction by the enzyme. Our data clearly show that initially oxidized pseudoazurin causes rapid further turnover by the enzyme to give a final product that we assign as all-ferric cytochrome cd(1) with nitrite bound to the d(1) heme (i.e. from which NO had dissociated). Pseudoazurin catalyzed this effect even when present at only one-tenth the stoichiometry of cytochrome cd(1). In contrast, redox-inert zinc pseudoazurin did not affect cd(1)-X, indicating a crucial role for electron movement between monomers or individual enzyme dimers rather than simply a protein-protein interaction. Furthermore, formation of cd(1)-X was, remarkably, accelerated by the presence of pseudoazurin, such that it occurred at a rate consistent with cd(1)-X being an intermediate in the catalytic cycle. It is clear that cytochrome cd(1) functions significantly differently in the presence of its two substrates, nitrite and electron donor protein, than in the presence of nitrite alone.     

Nitrite reduction: a ubiquitous function from a pre‐aerobic past

In eukaryotes, small amounts of nitrite confer cytoprotection against ischemia/reperfusion‐related tissue damage in vivo, possibly via reduction to nitric oxide (NO) and inhibition of mitochondrial function. Several hemeproteins are involved in this protective mechanism, starting with deoxyhemoglobin, which is capable of reducing nitrite. In facultative aerobic bacteria, such as Pseudomonas aeruginosa, nitrite is reduced to NO by specialized heme‐containing enzymes called cd₁ nitrite reductases. The details of their catalytic mechanism are summarized below, together with a hypothesis on the biological role of the unusual d₁‐heme, which, in the reduced state, shows unique properties (very high affinity for nitrite and exceptionally fast dissociation of NO). Our results support the idea that the nitrite‐based reactions of contemporary eukaryotes are a vestige of earlier bacterial biochemical pathways. The evidence that nitrite reductase activities of enzymes with different cellular roles and biochemical features still exist today highlights the importance of nitrite in cellular homeostasis.     

Time-resolved infrared spectroscopy reveals a stable ferric heme-NO intermediate in the reaction of Paracoccus pantotrophus cytochrome cd1 nitrite reductase with nitrite

Cytochrome cd(1) is a respiratory enzyme that catalyzes the physiological one-electron reduction of nitrite to nitric oxide. The enzyme is a dimer, each monomer containing one c-type cytochrome center and one active site d(1) heme. We present stopped-flow Fourier transform infrared data showing the formation of a stable ferric heme d(1)-NO complex (formally d(1)Fe(II)-NO(+)) as a product of the reaction between fully reduced Paracoccus pantotrophus cytochrome cd(1) and nitrite, in the absence of excess reductant. The Fe-(14)NO nu(NO) stretching mode is observed at 1913 cm(-1) with the corresponding Fe-(15)NO band at 1876 cm(-1). This d(1) heme-NO complex is still readily observed after 15 min. EPR and visible absorption spectroscopic data show that within 4 ms of the initiation of the reaction, nitrite is reduced at the d(1) heme, and a cFe(III) d(1)Fe(II)-NO complex is formed. Over the next 100 ms there is an electron redistribution within the enzyme to give a mixed species, 55% cFe(III) d(1)Fe(II)-NO and 45% cFe(II) d(1)Fe(II)-NO(+). No kinetically competent release of NO could be detected, indicating that at least one additional factor is required for product release by the enzyme. Implications for the mechanism of P. pantotrophus cytochrome cd(1) are discussed.     

NO production by Pseudomonas aeruginosa cd1 nitrite reductase

The structural and catalytic properties of Pseudomonas aeruginosa cd1 nitrite reductase, a key enzyme in bacterial denitrification, are reviewed in this paper. The mechanism of reduction of nitrite to NO is discussed in detail with special attention to the structural interpretation of function. The ability to stabilize negatively charged molecules, such as the substrate (nitrite) and other ligands (hydroxide and cyanide), is a key feature of catalysis in cd1NIRs. The positive potential in the active site is largely due to the presence of the two conserved distal histidines, which are involved in both substrate binding and product release.     

A random-sequential mechanism for nitrite binding and active site reduction in copper-containing nitrite reductase

The homotrimeric copper-containing nitrite reductase (NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of pH and nitrite on the turnover rate in the presence of three different electron donors at saturating concentrations were measured. The activity of NiR was also measured electrochemically by exploiting direct electron transfer to the enzyme immobilized on a graphite rotating disk electrode. In all cases, the steady-state kinetics fitted excellently to a random-sequential mechanism in which electron transfer from the type-1 to the type-2 site is rate-limiting. At low [NO(-)(2)] reduction of the type-2 site precedes nitrite binding, at high [NO(-)(2)] the reverse occurs. Below pH 6.5, the catalytic activity diminished at higher nitrite concentrations, in agreement with electron transfer being slower to the nitrite-bound type-2 site than to the water-bound type-2 site. Above pH 6.5, substrate activation is observed, in agreement with electron transfer to the nitrite-bound type-2 site being faster than electron transfer to the hydroxyl-bound type-2 site. To study the effect of slower electron transfer between the type-1 and type-2 site, NiR M150T was used. It has a type-1 site with a 125-mV higher midpoint potential and a 0.3-eV higher reorganization energy leading to an approximately 50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism.     

Internal electron transfer and structural dynamics of cd1 nitrite reductase revealed by laser CO photodissociation.

Laser photolysis techniques have been employed to investigate the internal electron transfer (eT) reaction within Pseudomonas aeruginosa nitrite reductase (Pa-NiR). We have measured the (d1--> c) internal eT rate for the wild-type protein and a site-directed mutant (Pa-NiR H327A) which has a substitution in the d1-heme binding pocket; we found the rate of eT to be fast, keT = 2.5 x 10(4) and 3.5 x 10(4) s-1 for the wild-type and mutant Pa-NiR, respectively. We also investigated the photodissociation of CO from the fully reduced proteins and observed microsecond first-order relaxations; these imply that upon breakage of the Fe2+-CO bond, both Pa-NiR and Pa-NiR H327A populate a nonequilibrium state which decays to the ground state with a complex time course that may be described by two exponential processes (k1 = 3 x 10(4) s-1 and k2 = 0.25 x 10(4) s-1). These relaxations do not have a kinetic difference spectrum characteristic of CO recombination, and therefore we conclude that Pa-NiR undergoes structural rearrangements upon dissociation of CO. The bimolecular rate of CO rebinding is 5 times faster in Pa-NiR H327A than in the wild-type enzyme (1.1 x 10(5) M-1 s-1 compared to 2 x 10(4) M-1 s-1), indicating that this mutation in the active site alters the CO diffusion properties of the protein, probably reducing steric hindrance. CO rebinding to the wild-type mixed valence enzyme (c3+d12+) which is very slow (k = 0.25 s-1) is proposed to be rate-limited by the c --> d1 internal eT event, involving the oxidized d1-heme which has a structure characteristic of the fully oxidized and partially oxidized Pa-NiR.     

Unexpected dependence on pH of NO release from Paracoccus pantotrophus cytochrome cd1

A previous study of nitrite reduction by Paracoccus pantotrophus cytochrome cd₁ at pH 7.0 identified early reaction intermediates. The c-heme rapidly oxidised and nitrite was reduced to NO at the d₁-heme. A slower equilibration of electrons followed, forming a stable complex assigned as 55% cFe(III)d₁Fe(II)-NO and 45% cFe(II)d₁Fe(II)-NO⁺. No catalytically competent NO release was observed. Here we show that at pH 6.0, a significant proportion of the enzyme undergoes turnover and releases NO. An early intermediate, which was previously overlooked, is also identified; enzyme immediately following product release is a candidate. However, even at pH 6.0 a considerable fraction of the enzyme remains bound to NO so another component is required for full product release. The kinetically stable product formed at the end of the reaction differs significantly at pH 6.0 and 7.0, as does its rate of formation; thus the reaction is critically dependent on pH.     

Mechanism of spinach chloroplast ferredoxin-dependent nitrite reductase: spectroscopic evidence for intermediate states

Nitrite reductases found in plants, algae, and cyanobacteria catalyze the six-electron reduction of nitrite to ammonia with reduced ferredoxin serving as the electron donor. They contain one siroheme and one [4Fe-4S] cluster, acting as separate one-electron carriers. Nitrite is thought to bind to the siroheme and to remain bound until its complete reduction to ammonia. In the present work the enzyme catalytic cycle, with ferredoxin reduced by photosystem 1 as an electron donor, has been studied by EPR and laser flash absorption spectroscopy. Substrate depletion during enzyme turnover, driven by a series of laser flashes, has been demonstrated. A complex of ferrous siroheme with NO, formed by two-electron reduction of the enzyme complex with nitrite, has been shown to be an intermediate in the enzyme catalytic cycle. The same complex can be formed by incubation of free oxidized nitrite reductase with an excess of nitrite and ascorbate. Hydroxylamine, another putative intermediate in the reduction of nitrite catalyzed by nitrite reductase, was found to react with oxidized nitrite reductase to produce the same ferrous siroheme-NO complex, with a characteristic formation time of about 13 min. The rate-limiting step for this reaction is probably hydroxylamine binding to the enzyme, with the conversion of hydroxylamine to NO at the enzyme active site likely being much faster.     

Nitric oxide modulates the catalytic activity of myeloperoxidase

Myeloperoxidase (MPO), an abundant protein in neutrophils, monocytes, and subpopulations of tissue macrophages, is believed to play a critical role in host defenses and inflammatory tissue injury. To perform these functions, an array of diffusible radicals and reactive oxidant species may be formed through oxidation reactions catalyzed at the heme center of the enzyme. Myeloperoxidase and inducible nitric-oxide synthase are both stored in and secreted from the primary granules of activated leukocytes, and nitric oxide (nitrogen monoxide; NO) reacts with the iron center of hemeproteins at near diffusion-controlled rates. We now demonstrate that NO modulates the catalytic activity of MPO through distinct mechanisms. NO binds to both ferric (Fe(III), the catalytically active species) and ferrous (Fe(II)) forms of MPO, generating stable low-spin six-coordinate complexes, MPO-Fe(III).NO and MPO-Fe(II).NO, respectively. These nitrosyl complexes were spectrally distinguishable by their Soret absorbance peak and visible spectra. Stopped-flow kinetic analyses indicated that NO binds reversibly to both Fe(III) and Fe(II) forms of MPO through simple one-step mechanisms. The association rate constant for NO binding to MPO-Fe(III) was comparable to that observed with other hemoproteins whose activities are thought to be modulated by NO in vivo. In stark contrast, the association rate constant for NO binding to the reduced form of MPO, MPO-Fe(II), was over an order of magnitude slower. Similarly, a 2-fold decrease was observed in the NO dissociation rate constant of the reduced versus native form of MPO. The lower NO association and dissociation rates observed suggest a remarkable conformational change that alters the affinity and accessibility of NO to the distal heme pocket of the enzyme following heme reduction. Incubation of NO with the active species of MPO (Fe(III) form) influenced peroxidase catalytic activity by dual mechanisms. Low levels of NO enhanced peroxidase activity through an effect on the rate-limiting step in catalysis, reduction of Compound II to the ground-state Fe(III) form. In contrast, higher levels of NO inhibited MPO catalysis through formation of the nitrosyl complex MPO-Fe(III)-NO. NO interaction with MPO may thus serve as a novel mechanism for modulating peroxidase catalytic activity, influencing the regulation of local inflammatory and infectious events in vivo.     

Proton-coupled structural changes upon binding of carbon monoxide to cytochrome cd1: a combined flash photolysis and X-ray crystallography study

We have investigated dynamic events after flash photolysis of CO from reduced cytochrome cd(1) nitrite reductase (NiR) from Paracoccus pantotrophus (formerly Thiosphaera pantotropha). Upon pulsed illumination of the cytochrome cd(1)-CO complex, at 460 nm, a rapid (<50 ns) absorbance change, attributed to dissociation of CO, was observed. This was followed by a biphasic rearrangement with rate constants of 1.7 x 10(4) and 2.5 x 10(3) s(-1) at pH 8.0. Both parts of the biphasic rearrangement phases displayed the same kinetic difference spectrum in the region of 400-660 nm. The slower of the two processes was accompanied by proton uptake from solution (0.5 proton per active site at pH 7.5-8.5). After photodissociation, the CO ligand recombined at a rate of 12 s(-1) (at 1 mM CO and pH 8.0), accompanied by proton release. The crystal structure of reduced cytochrome cd(1) in complex with CO was determined to a resolution of 1.57 A. The structure shows that CO binds to the iron of the d(1) heme in the active site. The ligation of the c heme is unchanged in the complex. A comparison of the structures of the reduced, unligated NiR and the NiR-CO complex indicates changes in the puckering of the d(1) heme as well as rearrangements in the hydrogen-bonding network and solvent organization in the substrate binding pocket at the d(1) heme. Since the CO ligand binds to heme d(1) and there are structural changes in the d(1) pocket upon CO binding, it is likely that the proton uptake or release observed after flash-induced CO dissociation is due to changes of the protonation state of groups in the active site. Such proton-coupled structural changes associated with ligand binding are likely to affect the redox potential of heme d(1) and may regulate the internal electron transfer from heme c to heme d(1).     

Quantum mechanical interpretation of nitrite reduction by cytochrome cd1 nitrite reductase from Paracoccus pantotrophus

The reduction of nitrite to nitric oxide in respiratory denitrification is catalyzed by a cytochrome cd(1) nitrite reductase in Paracoccus pantotrophus (formerly known as Thiosphaera pantotropha LMD 92.63). High-resolution structures are available for the fully oxidized [Fül?p, V., Moir, J. W., Ferguson, S. J., and Hajdu, J. (1995) Cell 81, 369-377; Baker, S. C., Saunders, N. F., Willis, A. C., Ferguson, S. J., Hajdu, J., and Fül?p, V. (1997) J. Mol. Biol. 269, 440-455] and fully reduced forms of this enzyme, as well as for various intermediates in its catalytic cycle [Williams, P. A., Fül?p, V., Garman, E. F., Saunders, N. F., Ferguson, S. J., and Hajdu, J. (1997) Nature 389, 406-412]. On the basis of these structures, quantum mechanical techniques (QM), including density functional methods (DFT), were combined with simulated annealing (SA) and molecular mechanics techniques (MM) to calculate the electronic distribution of molecular orbitals in the active site during catalysis. The results show likely trajectories for electrons, protons, substrates, and products in the process of nitrite reduction, and offer an interpretation of the reaction mechanism. The calculations indicate that the redox state of the d(1) heme and charges on two histidines in the active site orchestrate catalysis locally. Binding of nitrite to the reduced iron is followed by proton transfer from His345 and His388 to one of the oxygens of nitrite, creating a water molecule and an [Fe(II)-NO(+)] complex. Valence isomerization within this complex gives [Fe(III)-NO]. The release of NO from the ferric iron is influenced by the protonation state of His345 and His388, and by the orientation of NO on the d(1) heme. Return of Tyr25 to a hydrogen-bonding position between His345 and His388 facilitates product release, but a rebinding of Tyr25 to the oxidized iron may be bypassed in steady-state catalysis.     

Purification of cytochrome cd1 nitrite reductase from Pseudomonas stutzeri JM300 and reconstitution with native and synthetic heme d1.

Cytochrome cd1 nitrite reductase has been purified from Pseudomonas stutzeri strain JM 300. This enzyme appears to be a dimer with a subunit molecular mass of 54 kDa and its isoelectric point is determined to be 5.4. The N terminus of amino acid sequence has strong homology with that of nitrite reductase from P. aeruginosa. The apoprotein of this enzyme has been reconstituted with native and synthetic heme d1. The nitrite reductase activity measured by NO and N2O gas evolution can be restored to 82% of the activity of the original enzyme when the protein was reconstituted with the native heme d1 and to 77% of the activity when reconstituted with the synthetic heme d1. The absorption spectra of both reconstituted enzymes are essentially identical to that of the original nitrite reductase. These results further substantiate the novel dione structure of heme d1 as proposed. The loss of NO2- reducing activity in the absence of heme d1 and its restoration by addition of heme d1 provides further evidence that heme d1 plays a key role in the conversion of NO2- to NO and N2O.     

Interaction of nitric oxide with human heme oxygenase-1

NO and CO may complement each other as signaling molecules in some physiological situations. We have examined the binding of NO to human heme oxygenase-1 (hHO-1), an enzyme that oxidizes heme to biliverdin, CO, and free iron, to determine whether inhibition of hHO-1 by NO can contribute to the signaling interplay of NO and CO. An Fe(3+)-NO hHO-1-heme complex is formed with NO or the NO donors NOC9 or 2-(N,N-diethylamino)-diazenolate-2-oxide.sodium salt. Resonance Raman spectroscopy shows that ferric hHO-1-heme forms a 6-coordinated, low spin complex with NO. The nu(N-O) vibration of this complex detected by Fourier transform IR is only 4 cm(-1) lower than that of the corresponding metmyoglobin (met-Mb) complex but is broader, suggesting a greater degree of ligand conformational freedom. The Fe(3+)-NO complex of hHO-1 is much more stable than that of met-Mb. Stopped-flow studies indicate that k(on) for formation of the hHO-1-heme Fe(3+)-NO complex is approximately 50-times faster, and k(off) 10 times slower, than for met-Mb, resulting in K(d) = 1.4 microm for NO. NO thus binds 500-fold more tightly to ferric hHO-1-heme than to met-Mb. The hHO-1 mutations E29A, G139A, D140A, S142A, G143A, G143F, and K179A/R183A do not significantly diminish the tight binding of NO, indicating that NO binding is not highly sensitive to mutations of residues that normally stabilize the distal water ligand. As expected from the K(d) value, the enzyme is reversibly inhibited upon exposure to pathologically, and possibly physiologically, relevant concentrations of NO. Inhibition of hHO-1 by NO may contribute to the pleiotropic responses to NO and CO.     

Directing the mode of nitrite binding to a copper-containing nitrite reductase from Alcaligenes faecalis S-6: characterization of an active site isoleucine

Unlike the heme cd(1)-based nitrite reductase enzymes, the molecular mechanism of copper-containing nitrite reductases remains controversial. A key source of controversy is the productive binding mode of nitrite in the active site. To identify and characterize the molecular determinants associated with nitrite binding, we applied a combinatorial mutagenesis approach to generate a small library of six variants at position 257 in nitrite reductase from Alcaligenes faecalis S-6. The activities of these six variants span nearly two orders of magnitude with one variant, I257V, the only observed natural substitution for Ile257, showing greater activity than the native enzyme. High-resolution (> 1.8 A) nitrite-soaked crystal structures of these variants display different modes of nitrite binding that correlate well with the altered activities. These studies identify for the first time that the highly conserved Ile257 in the native enzyme is a key molecular determinant in directing a catalytically competent mode of nitrite binding in the active site. The O-coordinate bidentate binding mode of nitrite observed in native and mutant forms with high activity supports a catalytic model distinct from the heme cd(1) NiRs. (The atomic coordinates for I257V[NO(2)(-)], I257L[NO(2)(-)], I257A[NO(2)(-)], I257T[NO(2)(-)], I257M[NO(2)(-)] and I257G[NO(2)(-)] AfNiR have been deposited in the Protein Data Bank [PDB identification codes are listed in Table 2].)     

The reaction of Pseudomonas nitrite reductase and nitrite. A stopped-flow and EPR study

The reaction between reduced Pseudomonas nitrite reductase and nitrite has been studied by stopped-flow and rapid-freezing EPR spectroscopy. The interpretation of the kinetics at pH 8.0 is consistent with the following reaction mechanism (where k1 and k3 much greater than k2). [formula: see text] The bimolecular step (Step 1) is very fast, being lost in the dead time of a rapid mixing apparatus; the stoichiometry of the complex has been estimated to correspond to one NO2- molecule/heme d1. The final species is the fully reduced enzyme with NO bound to heme d1; and at all concentrations of nitrite, there is no evidence for dissociation of NO or for further reduction of NO to N2O. Step 2 is assigned to an internal electron transfer from heme c to reduced NO-bound heme d1 occurring with a rate constant of 1 s-1; this rate is comparable to the rate of internal electron transfer previously determined when reducing the oxidized enzyme with azurin or cytochrome c551. When heme d1 is NO-bound, the rate at which heme c can accept electrons from ascorbate is remarkably increased as compared to the oxidized enzyme, suggesting an increase in the redox potential of the latter heme.