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In nitrite reductase (cd(1) NIR), the c-heme mediates electron transfer to the catalytic d(1)-heme where nitrite (NO(2)(-)) is reduced to nitric oxide (NO). An interesting feature of this enzyme is the relative lability of the reaction product NO bound to the d(1)-heme. Marked differences in the c- to d(1)-heme electron-transfer rates were reported for cd(1) NIRs from different sources, such as Pseudomonas stutzeri (P. stutzeri) and Pseudomonas aeruginosa (P. aeruginosa). The three-dimensional structure of the P. aeruginosa enzyme has been determined, but that of the P. stutzeri enzyme is still unknown. The difference in electron transfer rates prompted a comparison of the structural properties of the d(1)-heme pocket of P. stutzeri cd(1) NIR with those of the P. aeruginosa wild type enzyme (WT) and its Y10F using their nitrosyl d(1)-heme complexes. We applied high field pulse electron paramagnetic resonance (EPR) techniques that detect nuclear spins in the close environment of the spin bearing Fe(II)-NO entity. We observed similarities in the rhombic g-tensor and detected a proximal histidine ligand with (14)N hyperfine and quadrupole interactions also similar to those of P. aeruginosa WT and Y10F mutant complexes. In contrast, we also observed significant differences in the H-bond network involving the NO ligand and a larger solvent accessibility for P. stutzeri attributed to the absence of this tyrosine residue. For P. aeruginosa, cd(1) NIR domain swapping allows Tyr(10) to become H-bonded to the bound NO substrate. These findings support a previous suggestion that the large difference in the c- to d(1)-heme electron transfer rates between the two enzymes is related to solvent accessibility of their d(1)-heme pockets.

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.

W-band (95 GHz) HYSCORE and pulse ENDOR are used to characterize the nitrosyl d(1) heme complex (d(1)NO) of cd(1) nitrite reductase from Pseudomonas aeruginosa in the wild type and the Y10F mutant. The spectra and the derived (14)N hyperfine and quadrupole interactions were found to be the same for wt and Y10F. This suggests that Tyr10 does not influence the NO ligand orientation in the reduced state in solution. This study is the first application of HYSCORE at high fields and shows its potential for characterizing low gamma nuclei with large hyperfine couplings.

The cd(1) nitrite reductases, which catalyze the reduction of nitrite to nitric oxide, are homodimers of 60 kDa subunits, each containing one heme-c and one heme-d(1). Heme-c is the electron entry site, whereas heme-d(1) constitutes the catalytic center. The 3D structure of Pseudomonas aeruginosa nitrite reductase has been determined in both fully oxidized and reduced states. Intramolecular electron transfer (ET), between c and d(1) hemes is an essential step in the catalytic cycle. In earlier studies of the Pseudomonas stutzeri enzyme, we observed that a marked negative cooperativity is controlling this internal ET step. In this study we have investigated the internal ET in the wild-type and His369Ala mutant of P. aeruginosa nitrite reductases and have observed similar cooperativity to that of the Pseudomonas stutzeri enzyme. Heme-c was initially reduced, in an essentially diffusion-controlled bimolecular process, followed by unimolecular electron equilibration between the c and d(1) hemes (k(ET) = 4.3 s(-1) and K = 1.4 at 298 K, pH 7.0). In the case of the mutant, the latter ET rate was faster by almost one order of magnitude. Moreover, the internal ET rate dropped (by approximately 30-fold) as the level of reduction increased in both the WT and the His mutant. Equilibrium standard enthalpy and entropy changes and activation parameters of this ET process were determined. We concluded that negative cooperativity is a common feature among the cd(1) nitrite reductases, and we discuss this control based on the available 3D structure of the wild-type and the H369A mutant, in the reduced and oxidized states.

In the denitrification pathway, Pseudomonas aeruginosa cytochrome cd1 nitrite reductase catalyzes the reduction of nitrite to nitric oxide; in vitro, this enzyme is also competent in the reduction of O2 to 2H2O. In this article, we present a comparative kinetic study of the O2 reaction in the wild-type nitrite reductase and in three site-directed mutants (Tyr10-->Phe, His369-->Ala and His327-->Ala/His369-->Ala) of the amino acid residues close to the d1 heme on the distal side. The results clearly indicate that His369 is the key residue in the control of reactivity, as its substitution with Ala, previously shown to affect the reduction of nitrite, also impairs the reaction with O2, affecting both the properties and lifespan of the intermediate species. Our findings allow the presentation of an overall picture for the reactivity of cytochrome cd1 nitrite reductase and extend our previous conclusion that the conserved distal histidines are essential for the binding to reduced d1 heme of different anions, whether a substrate such as nitrite, a ligand such as cyanide, or an intermediate in the O2 reduction. Moreover, we propose that His369 also exerts a protective role against degradation of the d1 heme, by preventing the formation and adverse effects of the reactive O2 species (never present in significant amounts in wild-type cytochrome cd1 nitrite reductase), a finding with clear physiological implications.

A model-free analysis of Pseudomonas aeruginosa ferricytochrome c551 dynamics based on 15N R1, 15N R2, and {1H}-15N heteronuclear nuclear Overhauser effect data is reported. The protein backbone is highly rigid (2>=0.924±0.005) and displays little variation in picosecond–nanosecond time scale dynamics over the structure. The loop structure containing the axial methionine ligand (loop 3) displays anomalous rigidity, which is attributed to its high proline content. Also reported are protection factors calculated from hydrogen-exchange rates. These data reveal that loop 3 residues, including the axial methionine, are protected from exchange as a result of long-range hydrogen-bonding interactions. These results are contrasted with data reported for Saccharomyces cerevisiae iso-1-ferricytochrome c, which displays higher overall flexibility (2>=0.80±0.07), greater variation of dynamics as a function of structure, and low protection factors for loop 3. This analysis reveals that heme proteins with similar functions and topologies may display diverse dynamical properties. Electronic supplementary material is available if you access this article at http://dx.doi.org/10.1007/s00775-002-0401-z. On that page (frame on the left side), a link takes you directly to the supplementary material. [ABSTRACT FROM AUTHOR]

Cytochrome c551 from Pseudomonas aeruginosa is a monomeric redox protein of 82 amino-acid residues, involved in dissimilative denitrification as the physiological electron donor of cd1 nitrite reductase. The distribution of charged residues on the surface of c551 is very anisotropic: one side is richer in acidic residues whereas the other shows a ring of positive side chains, mainly lysines, located at the border of an hydrophobic patch which surrounds the heme crevice. In order to map in cytochrome c551 the surface involved in electron transfer, we have introduced specific mutations in three residues belonging to the hydrophobic patch, namely Val23→Asp, Pro58→Ala and Ile59→Glu. The effect of these mutations was analyzed studying both the self-exchange rate and the electron-transfer activity towards P. aeruginosa cd1 nitrite reductase, the physiological partner and P. aeruginosa azurin, a copper protein often used as a model redox partner in vitro. Our results show that introduction of a negative charge in the hydrophobic patch severely hampers both homonuclear and heteronuclear electron transfer. [ABSTRACT FROM AUTHOR]

Reports on the creation of an electric dipole in the hydrophobic patch of azurin from Pseudomonas aeruginosa. Effect of the dipole on the electron-transfer properties of azurin; Proposal of electron transfer as the physiological redox partners cytochrome c[sub 551] and nitrite reductase.

Cytochrome cd 1 nitrite reductase (cd 1 NIR) is a haem-containing enzyme responsible for the reduction of nitrite into nitric oxide (NO), a key step in the bacterial anaerobic respiratory process of denitrification. The active site of cytochrome cd 1 contains the unique d 1-haem cofactor, from which NO must be released. In general, reduced haems bind NO tightly relative to oxidised haems. The mechanism of NO release employed by cd 1 NiRs has been recently outlined, highlighting the essential role of the peculiar d 1-haem, whose presence in these enzymes was largely unexplained. Briefly, we have shown that cd 1 NiRs can release quite rapidly the NO bound to the reduced d 1 haem. This finding challenged the classical idea that NO is often an “irreversible” inhibitor of reduced hemoproteins and prompted new studies on the reactivity of ferrous d 1 haem with NO, indicating that the d 1 haem has evolved to have low affinity for NO, as compared to other ferrous haems. [Copyright &y& Elsevier]

Considerable progress was made over the last few years in understanding the mechanism of folding of cytochrome c551, a small acidic hemeprotein from Pseudomonas aeruginosa. Comparison of our results with those obtained by others on horse heart cytochrome c allows to draw some general conclusions on the structural features that are common determinants in the folding of members of the cytochrome c family. [Copyright &y& Elsevier]

A model-free analysis of Pseudomonas aeruginosa ferricytochrome c551 dynamics based on 15N R1, 15N R2, and {1H}-15N heteronuclear nuclear Overhauser effect data is reported. The protein backbone is highly rigid (=0.924±0.005) and displays little variation in picosecond–nanosecond time scale dynamics over the structure. The loop structure containing the axial methionine ligand (loop 3) displays anomalous rigidity, which is attributed to its high proline content. Also reported are protection factors calculated from hydrogen-exchange rates. These data reveal that loop 3 residues, including the axial methionine, are protected from exchange as a result of long-range hydrogen-bonding interactions. These results are contrasted with data reported for Saccharomyces cerevisiae iso-1-ferricytochrome c, which displays higher overall flexibility (=0.80±0.07), greater variation of dynamics as a function of structure, and low protection factors for loop 3. This analysis reveals that heme proteins with similar functions and topologies may display diverse dynamical properties. Electronic supplementary material is available if you access this article at http://dx.doi.org/10.1007/s00775-002-0401-z. On that page (frame on the left side), a link takes you directly to the supplementary material. [ABSTRACT FROM AUTHOR]