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.     

Paracoccus pantotrophus NapC can reductively activate cytochrome cd1 nitrite reductase

The oxidized "as isolated" form of Paracoccus pantotrophus cytochrome cd1 nitrite reductase has a bis-histidinyl coordinated c heme and a histidine/tyrosine coordinated d1 heme. This form of the enzyme has previously been shown to be kinetically incompetent. Upon reduction, the coordination of both hemes changes and the enzyme is kinetically activated. Here, we show that P. pantotrophus NapC, a tetraheme c-type cytochrome belonging to a large family of such proteins, is capable of reducing, and hence activating, "as isolated" cytochrome cd1. NapC is the first protein from P. pantotrophus identified as being capable of this activation step and, given the periplasmic co-location and co-expression of the two proteins, is a strong candidate to be a physiological activation partner.     

Oxidase reaction of cytochrome cd(1) from Paracoccus pantotrophus

Cytochrome cd(1) (cd(1)NIR) from Paracoccus pantotrophus, which is both a nitrite reductase and an oxidase, was reduced by ascorbate plus hexaamineruthenium(III) chloride on a relatively slow time scale (hours required for complete reduction). Visible absorption spectroscopy showed that mixing of ascorbate-reduced enzyme with oxygen at pH = 6.0 resulted in the rapid oxidation of both types of heme center in the enzyme with a linear dependence on oxygen concentration. Subsequent changes on a longer time scale reflected the formation and decay of partially reduced oxygen species bound to the d(1) heme iron. Parallel freeze-quench experiments allowed the X-band electron paramagnetic resonance (EPR) spectrum of the enzyme to be recorded at various times after mixing with oxygen. On the same millisecond time scale that simultaneous oxidation of both heme centers was seen in the optical experiments, two new EPR signals were observed. Both of these are assigned to oxidized heme c and resemble signals from the cytochrome c domain of a "semi-apo" form of the enzyme for which histidine/methionine coordination was demonstrated spectroscopically. These observations suggests that structural changes take around the heme c center that lead to either histidine/methionine axial ligation or a different stereochemistry of bis-histidine axial ligation than that found in the as prepared enzyme. At this stage in the reaction no EPR signal could be ascribed to Fe(III) d(1) heme. Rather, a radical species, which is tentatively assigned to an amino acid radical proximal to the d(1) heme iron in the Fe(IV)-oxo state, was seen. The kinetics of decay of this radical species match the generation of a new form of the Fe(III) d(1) heme, probably representing an OH(-)-bound species. This sequence of events is interpreted in terms of a concerted two-electron reduction of oxygen to bound peroxide, which is immediately cleaved to yield water and an Fe(IV)-oxo species plus the radical. Two electrons from ascorbate are subsequently transferred to the d(1) heme active site via heme c to reduce both the radical and the Fe(IV)-oxo species to Fe(III)-OH(-) for completion of a catalytic cycle.     

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.     

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.     

The Structure of an alternative form of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase

Cytochrome cd(1) nitrite reductase is a bifunctional enzyme, which can catalyze the 1-electron reduction of nitrite to nitric oxide and the 4-electron reduction of dioxygen to water. Here we describe the structure of reduced nitrite reductase, crystallized under anaerobic conditions. The structure reveals substantial domain rearrangements with the c domain rotated by 60 degrees and shifted by approximately 20 A compared with previously known structures from crystals grown under oxidizing conditions. This alternative conformation gives rise to different electron transfer routes between the c and d(1) domains and points to the involvement of elements of very large structural changes in the function in this enzyme. In the present structure, the c heme has a His-69/Met-106 ligation, and this ligation does not change upon oxidation in the crystal. The d(1) heme is penta-coordinated, and the d(1) iron is displaced from the heme plane by 0.5 A toward the proximal ligand, His-200. After oxidation, the iron moves into the d(1) heme plane. A surprising finding is that although reduced nitrite reductase can be readily oxidized by dioxygen in the new crystal, it cannot turnover with its other substrate, nitrite. The results suggest that the rearrangement of the domains affects catalysis and substrate selectivity.     

X-ray crystallographic study of cyanide binding provides insights into the structure-function relationship for cytochrome cd1 nitrite reductase from Paracoccus pantotrophus

We present a 1.59-A resolution crystal structure of reduced Paracoccus pantotrophus cytochrome cd(1) with cyanide bound to the d(1) heme and His/Met coordination of the c heme. Fe-C-N bond angles are 146 degrees for the A subunit and 164 degrees for the B subunit of the dimer. The nitrogen atom of bound cyanide is within hydrogen bonding distance of His(345) and His(388) and either a water molecule in subunit A or Tyr(25) in subunit B. The ferrous heme-cyanide complex is unusually stable (K(d) approximately 10(-6) m); we propose that this reflects both the design of the specialized d(1) heme ring and a general feature of anion reductases with active site heme. Oxidation of crystals of reduced, cyanide-bound, cytochrome cd(1) results in loss of cyanide and return to the native structure with Tyr(25) as a ligand to the d(1) heme iron and switching to His/His coordination at the c-type heme. No reason for unusually weak binding of cyanide to the ferric state can be identified; rather it is argued that the protein is designed such that a chelate-based effect drives displacement by tyrosine of cyanide or a weaker ligand, like reaction product nitric oxide, from the ferric d(1) heme.     

Structure and kinetic properties of Paracoccus pantotrophus cytochrome cd1 nitrite reductase with the d1 heme active site ligand tyrosine 25 replaced by serine

The 1.4-A crystal structure of the oxidized state of a Y25S variant of cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus is described. It shows that loss of Tyr(25), a ligand via its hydroxy group to the iron of the d(1) heme in the oxidized (as prepared) wild-type enzyme, does not result in a switch at the c heme of the unusual bishistidinyl coordination to the histidine/methionine coordination seen in other conformations of the enzyme. The Ser(25) side chain is seen in two positions in the d(1) heme pocket with relative occupancies of approximately 7:3, but in neither case is the hydroxy group bound to the iron atom; instead, a sulfate ion from the crystallization solution is bound between the Ser(25) side chain and the heme iron. Unlike the wild-type enzyme, the Y25S mutant is active as a reductase toward nitrite, oxygen, and hydroxylamine without a reductive activation step. It is concluded that Tyr(25) is not essential for catalysis of reduction of any substrate, but that the requirement for activation by reduction of the wild-type enzyme is related to a requirement to drive the dissociation of this residue from the active site. The Y25S protein retains the d(1) heme less well than the wild-type protein, suggesting that the tyrosine residue has a role in stabilizing the binding of this cofactor.     

A novel conformer of oxidized Paracoccus pantotrophus cytochrome cd(1) observed by freeze-quench NIR-MCD spectroscopy

Paracoccus pantotrophus cytochrome cd(1) is a physiological nitrite reductase and an in vitro hydroxylamine reductase. The oxidised "as isolated" form of the enzyme has bis-histidinyl coordinated c-heme and upon reduction its coordination changes to histidine/methionine. Following treatment of reduced enzyme with hydroxylamine, a novel, oxidised, conformer of the enzyme is obtained. We have devised protocols for freeze-quench near-ir-MCD spectroscopy that have allowed us to establish unequivocally the c-heme coordination of this species as His/Met. Thus it is shown that the catalytically competent, hydroxylamine reoxidised, form of P. pantotrophus cytochrome cd(1) has different axial ligands to the c-heme than "as isolated" enzyme.     

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.     

Activation of the cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. Reaction of oxidized enzyme with substrate drives a ligand switch at heme c

Cytochromes cd(1) are dimeric bacterial nitrite reductases, which contain two hemes per monomer. On reduction of both hemes, the distal ligand of heme d(1) dissociates, creating a vacant coordination site accessible to substrate. Heme c, which transfers electrons from donor proteins into the active site, has histidine/methionine ligands except in the oxidized enzyme from Paracoccus pantotrophus where both ligands are histidine. During reduction of this enzyme, Tyr(25) dissociates from the distal side of heme d(1), and one heme c ligand is replaced by methionine. Activity is associated with histidine/methionine coordination at heme c, and it is believed that P. pantotrophus cytochrome cd(1) is unreactive toward substrate without reductive activation. However, we report here that the oxidized enzyme will react with nitrite to yield a novel species in which heme d(1) is EPR-silent. Magnetic circular dichroism studies indicate that heme d(1) is low-spin Fe(III) but EPR-silent as a result of spin coupling to a radical species formed during the reaction with nitrite. This reaction drives the switch to histidine/methionine ligation at Fe(III) heme c. Thus the enzyme is activated by exposure to its physiological substrate without the necessity of passing through the reduced state. This reactivity toward nitrite is also observed for oxidized cytochrome cd(1) from Pseudomonas stutzeri suggesting a more general involvement of the EPR-silent Fe(III) heme d(1) species in nitrite reduction.     

Aerobic dissimilatory reduction of nitrite by cells of Paracoccus denitrificans: the role of nitric oxide

With anaerobically grown cells of Paracoccus denitrificans it was previously found (Kučera, I. and Dadák, V. (1983) Biochem. Biophys. Res. Commun. 117, 252–258) that, in the presence of an uncoupler, nitrite as terminal acceptor was preferred to oxygen, the consumption of which was simultaneously inhibited. In the present study it is shown that besides an increased inhibition of terminal oxidases brought about by NO₂⁻ anion another potent inhibitor originating in the course of nitrite reductase reaction affects the division of electron flow between oxygen and nitrite. The inhibitor, the creation of which is accompanied by the aerobic nitrite reduction, is formed, even in the absence of an uncoupler, only as a result of a slight inhibition of oxygen respiration exhibited by hydroxylamine addition. From the comparison of the inhibitory effect of the intermediate on aerobically grown cells and membrane vesicles derived from them, it was proved that at neutral pH this substance does not carry an electric charge. A complex absorbing at 563 nm was formed due to the interaction of the inhibitor (generated from nitrite in the presence of uncoupler) with ferricytochrome c from bovine heart. From these findings we were led to conclude that it was most probably nitric oxide (NO).     

Isolation of Paracoccus denitrificans cytochrome cd1: comparative kinetics with other nitrite reductases

The dissimilatory nitrite reductase of the cytochrome cd₁ type was purified from Paracoccus denitrificans (ATCC 13543) by a novel procedure that avoided conventional ion-exchange techniques. The characterization of this enzyme was extended to include amino acid composition, extinction coefficients, and kinetic properties not previously reported. Cytochromes cd₁ from Alicaligenes faecalis and Pseudomonas aeruginosa were also isolated and assayed with electron donor proteins. The enzymes from all three sources were shown to obey the same integrated rate law. Cross-reactivities were measured in which a reduced donor protein from one strain was assayed with cytochrome cd₁ from another strain using nitrite as ultimate acceptor. Donors included c-type cytochromes and azurins. In general, the enzymes showed specificity for a donor from the same strain; interspecies cross-reactions were typically slower on the order of 10-fold than corresponding native rates. Notable exceptions were Paracoccus cytochrome cd₁, which alone reacted with eukaryotic horse cytochrome c at appreciable rates, and the Pseudomonas cd₁-Alcaligenesc554 reaction, which was 4-fold faster than the native Alcaligenes cd₁-Alcaligenesc554 reaction. For all three enzymes, competitive kinetics were measured in which the alternative substrates, nitrite and oxygen, competed for enzyme in the same assay. It was found that the competitive kinetics were dominated by nonenzymatic reactions involving an enzyme product, nitric oxide.     

Very early reaction intermediates detected by microsecond time scale kinetics of cytochrome cd1-catalyzed reduction of nitrite

Paracoccus pantotrophus cytochrome cd(1) is a nitrite reductase found in the periplasm of many denitrifying bacteria. It catalyzes the reduction of nitrite to nitric oxide during the denitrification part of the biological nitrogen cycle. Previous studies of early millisecond intermediates in the nitrite reduction reaction have shown, by comparison with pH 7.0, that at the optimum pH, approximately pH 6, the earliest intermediates were lost in the dead time of the instrument. Access to early time points (approximately 100 micros) through use of an ultra-rapid mixing device has identified a spectroscopically novel intermediate, assigned as the Michaelis complex, formed from reaction of fully reduced enzyme with nitrite. Spectroscopic observation of the subsequent transformation of this species has provided data that demand reappraisal of the general belief that the two subunits of the enzyme function independently.     

Structure of the bound dioxygen species in the cytochrome oxidase reaction of cytochrome cd1 nitrite reductase

Reduction of dioxygen to water is a key process in aerobic life, but atomic details of this reaction have been elusive because of difficulties in observing active oxygen intermediates by crystallography. Cytochrome cd(1) is a bifunctional enzyme, capable of catalyzing the one-electron reduction of nitrite to nitric oxide, and the four-electron reduction of dioxygen to water. The latter is a cytochrome oxidase reaction. Here we describe the structure of an active dioxygen species in the enzyme captured by cryo-trapping. The productive binding mode of dioxygen in the active site is very similar to that of nitrite and suggests that the catalytic mechanisms of oxygen reduction and nitrite reduction are closely related. This finding has implications to the understanding of the evolution of oxygen-reducing enzymes. Comparison of the dioxygen complex to complexes of cytochrome cd(1) with stable diatomic ligands shows that nitric oxide and cyanide bind in a similar bent conformation to the iron as dioxygen whereas carbon monoxide forms a linear complex. The significance of these differences is discussed.     

Activation and catalysis of the di-heme cytochrome c peroxidase from Paracoccus pantotrophus

Bacterial cytochrome c peroxidases contain an electron transferring (E) heme domain and a peroxidatic (P) heme domain. All but one of these enzymes are isolated in an inactive oxidized state and require reduction of the E heme by a small redox donor protein in order to activate the P heme. Here we present the structures of the inactive oxidized and active mixed valence enzyme from Paracoccus pantotrophus. Chain flexibility in the former, as expressed by the crystallographic temperature factors, is strikingly distributed in certain loop regions, and these coincide with the regions of conformational change that occur in forming the active mixed valence enzyme. On the basis of these changes, we postulate a series of events that occur to link the trigger of the electron entering the E heme from either pseudoazurin or cytochrome c(550) and the dissociation of a coordinating histidine at the P heme, which allows substrate access.     

Solubilization and resolution of the membrane-bound nitrite reductase from Paracoccus halodenitrificans into nitrite and nitric oxide reductases

Membranes prepared from Paracoccus halodenitrificans reduced nitrite or nitric oxide to nitrous oxide. Extraction of these membranes with the detergent CHAPSO [3-(3-cholamidopropyldimethylammonio)-1-(2-hydroxy-1-propanesulfonate)], followed by ammonium sulfate fractionation of the solubilized proteins, resulted in the separation of nitrite and nitric oxide reductase activities. The fraction containing nitrite reductase activity spectrally resembled a cd-type cytochrome. Several cytochromes were detected in the nitric oxide reductase fraction. Which, if any, of these cytochromes is associated with the reduction of nitric oxide is not clear at this time.Abbreviations PMS phenazine methosulfate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - CHAPSO 3-(3-cholamidopropyl-dimethylammonio)-1-(2-hydroxy-1-propanesulfonate) - NH buffer 150 mM NaCl-50 mM - HEPES pH 7.5; octylglucoside, octyl--d glucopyranoside - NIR intrite reductase (nitrite to nitric oxide) - NOR nitric oxide reductase (nitric oxide to nitrous oxide)     

Identification of nitric oxide and nitrous oxide as products of nitrite reduction by Pseudomonas cytochrome oxidase (nitrate reductase)

The cytosol fraction of rat adrenocortical tissue contains comparatively high levels of two prostaglandin metabolizing enzymes. The first, prostaglandin-9-ketoreductase, utilizes NADPH more effectively than NADH as cofactor, is inhibited by NADP, and exhibits an apparent K_m of 304 μM for PGE₁. 15-hydroxyprostaglandin dehydrogenase, tentatively identified as the type II NADP-dependent isozyme, is inhibited by NADPH but not NADH, and exhibits an apparent K_m of 157 μM when PGE₁ is used as substrate. Changes in specific activities of the two enzymes following ACTH, hypophysectomy, or dexamethasone treatment are inconclusive in defining a chronic regulatory role for adrenocorticotropin.     

Evidence for water as the product for oxygen reduction by cytochrome cd.

Evidence is presented that water is the final product of electron donation to molecular oxygen by cytochrome $\text{cd}$ from $\text{Paracoccus}\text{denitrificans}$ when ferrocytochrome $\text{c}$ acts as donor to $\text{cd}$. Negative evidence for the accumulation of superoxide and peroxide was obtained by rate effect experiments in the presence of superoxide dismutase, catalase, and peroxidase. Positive evidence for water was obtained by showing a 4 to 1 stoichiometric balance for rates of electron acceptance from ferrocytochrome $\text{c}$ to rates of donation to molecular oxygen.