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. 2001 Feb 27;98(5):2232-7.
doi: 10.1073/pnas.041365298.

The nitrite reductase from Pseudomonas aeruginosa: essential role of two active-site histidines in the catalytic and structural properties

F Cutruzzola  1 , K Brown, E K Wilson, A Bellelli, M Arese, M Tegoni, C Cambillau, M Brunori
Affiliations

The nitrite reductase from Pseudomonas aeruginosa: essential role of two active-site histidines in the catalytic and structural properties

F Cutruzzola et al. Proc Natl Acad Sci U S A. .

Abstract

Cd(1) nitrite reductase catalyzes the conversion of nitrite to NO in denitrifying bacteria. Reduction of the substrate occurs at the d(1)-heme site, which faces on the distal side some residues thought to be essential for substrate binding and catalysis. We report the results obtained by mutating to Ala the two invariant active site histidines, His-327 and His-369, of the enzyme from Pseudomonas aeruginosa. Both mutants have lost nitrite reductase activity but maintain the ability to reduce O(2) to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form, possibly because the productive displacement of NO from the ferric d(1)-heme iron is impaired. Moreover, the two distal His play different roles in catalysis; His-369 is absolutely essential for the stability of the Michaelis complex. The structures of both mutants show (i) the new side chain in the active site, (ii) a loss of density of Tyr-10, which slipped away with the N-terminal arm, and (iii) a large topological change in the whole c-heme domain, which is displaced 20 A from the position occupied in the wild-type enzyme. We conclude that the two invariant His play a crucial role in the activity and the structural organization of cd(1) nitrite reductase from P. aeruginosa.

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Figures

Figure 1
Figure 1
The active site of cd1 NIR from P. aeruginosa (Pa-NIR). The model of the d1-heme pocket of the wt reduced enzyme complexed with nitrite is shown in A. The stereochemistry of NO2− was simulated starting from the coordinates of the NO adduct of reduced Pa-NiR (5). Among the key amino acid side chains shown here, notice that Tyr-10 comes from the other monomer (identified as sub. B), as a result of a domain swapping across the 2-fold axis of the homodimer (4). The 3D structure of the d1-heme pocket of the two mutants in the oxidized state is shown in the same orientation in B (for H369A) and C (for H327A). The Fo−Fc Sigma A negative electron density map is also represented at the place of the missing side chain; this map is contoured at −3σ for B and −4σ for C.
Figure 2
Figure 2
Static and transient optical spectra of wt and mutants Pa-NIR. (Upper) Absolute spectra of the oxidized (bold line), reduced (thin line), and reduced NO (dotted line) derivatives for the wt NIR (Left), mutant H327A (Center), and mutant H369A (Right). (Lower) Time evolution of the kinetic difference spectra observed for the same proteins, after the reduced enzyme is mixed with nitrite anaerobically. The two sets of difference spectra for each protein refer to experiments carried out at the lowest (10 μM; A, C, and E) and the highest (0.15 or 0.5 mM; B, D, and F) nitrite concentrations. The arrow indicates the direction of the time course, from 6 ms to 245 s. To better follow the spectral evolution with time, the difference spectra at selected times (6 ms and 1, 25, and 180 s) are drawn as thick lines. The insets show the time course as followed at the maximum of reduced d1-heme (462 nm for the wt and 472 nm for the two mutants), fitted to two or three exponentials (continuous lines). Kinetic experiments were carried out in 50 mM phosphate buffer (pH 8.0) and 25°C.
Figure 3
Figure 3
A proposed scheme for the reaction mechanism of cd1 NIR with NO2−. When nitrite is mixed with the reduced enzyme (species 1), the formation of the Michaelis complex (species 2) is followed by very rapid formation of NO, involving bond breaking and loss of a hydroxide ion; yet this process is assumed to be reversible. The resulting mixed-valence species 3 or 4 (c+2d1+3 NO or c+2d1+2 NO+) may dissociate NO and be reduced to species 1 (via 6 and 7) to enter a new productive cycle; however, in vitro it seems to be progressively inhibited to a dead-end state with NO bound to the ferrous d1-heme (species 8). Species 1–5 equilibrate rapidly [see also George et al. (20)], accounting for a fraction of oxidized c-heme formed during the dead time of the stopped flow. The relative population of each species depends on experimental conditions, such as pH and concentration of substrate and reductant, and it may even differ in NIR from different species. The Michaelis complex, formed rapidly even at low nitrite concentrations (e.g., 100 μM), accounts for considerably less than 100% of the enzyme; therefore the bimolecular rate constant is fast, but the affinity is lower than previously suggested (24). Nevertheless, our kinetic data are consistent with the value of Km = 6 μM NO2−, which was independently determined (not shown). Species 6 builds up slowly (k = 2 s−1) and incompletely under our experimental conditions; the internal redox equilibrium between species 6 and 7 is assigned a rate constant of 0.3 s−1. Because it is well established that the electron accepting site is the c-heme, only species 5 and 7 can be reduced with the use of ascorbate, azurin, or cytochrome c551. In the scheme, there are two paths leading to c+2d1+2 NO, the dead-end species 8: either by reaction of the reduced enzyme with NO or by reduction of species 5 by external reductants. In P. pantotrotrophus NiR, species 5 forms completely and instantaneously at [NO2−] = 0.2–5.0 mM; thereafter it decays to species 4 at 38 s−1, as shown by George et al. (20); it is possible that a similar reequilibration also occurs in Pa-NIR, but if so, it is lost in the dead time.
Figure 4
Figure 4
Crystallographic structure of the two mutants of Pa-NiR. (A) Top view of the mutant proteins superimposed on the wt enzyme. Color code for the c-heme domains: dark green, H327A; light green, H369A; red, wt. It is evident that the c-heme domains glide away in a new configuration, which is almost the same for the two mutants. Notice also that the 3D structure of the d1-heme domain (gray) is identical for the three proteins. (B) Side view of the three structures (same color code); only one monomer is shown for the sake of clarity.
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