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Crystal Structure of Naphthalene Dioxygenase: Side-on Binding of Dioxygen to Iron

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Science  14 Feb 2003:
Vol. 299, Issue 5609, pp. 1039-1042
DOI: 10.1126/science.1078020

Abstract

Binding of oxygen to iron is exploited in several biological and chemical processes. Although computational and spectroscopic results have suggested side-on binding, only end-on binding of oxygen to iron has been observed in crystal structures. We have determined structures of naphthalene dioxygenase that show a molecular oxygen species bound to the mononuclear iron in a side-on fashion. In a complex with substrate and dioxygen, the dioxygen molecule is lined up for an attack on the double bond of the aromatic substrate. The structures reported here provide the basis for a reaction mechanism and for the high stereospecificity of the reaction catalyzed by naphthalene dioxygenase.

Oxygenases that catalyze the addition of molecular oxygen to organic substrates play a pivotal role in diverse areas such as drug metabolism and the biodegradation of environmental pollutants. Oxygen activation by cytochrome(s) P450 (1–6) and diiron enzymes such as methane monooxygenase (MMO) (7–10) have been studied in detail. In contrast, less is known about bacterial Rieske non-heme iron dioxygenases (RDOs) that catalyze the stereospecific addition of dioxygen to aromatic hydrocarbons (11). The reaction products are chiral arenecis-dihydrodiols that are of current interest in enantioselective synthesis (12).

Naphthalene dioxygenase (NDO) from Pseudomonas sp., the only RDO for which a crystal structure is known (13), oxidizes naphthalene tocis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (14). The enzyme has an α3β3composition, and each α subunit contains a Rieske [2Fe-2S] center and mononuclear iron at the active site. Electrons from the reduced form of nicotinamide adenine dinucleotide (NADH) are transferred to NDO via an iron-sulfur flavoprotein (15) and a Rieske ferredoxin (16). The subsequent steps lead to oxygen activation and the formation of naphthalene cis-dihydrodiol. Although several hypotheses have been advanced to account for oxygen activation and catalysis by RDOs, the reaction mechanism remains elusive (17–21). To understand the molecular basis for the reaction, we have formed complexes of NDO with substrates, oxygen, substrate plus oxygen, and product and have determined their structures by x-ray crystallography (Table 1).

Table 1

Data collection and refinement details of the four structures reported. Numbers in parentheses indicate the last resolution shell. All crystallographic refinement was carried out with the program Refmac5.

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Substrate binding was achieved by soaking crystals of NDO in ethanol solutions of the substrates as described (22). Indole (22) and naphthalene (Fig. 1A) bind in an elongated cleft, with the carbon atoms to be hydroxylated at a distance of about 4 Å from the ferrous iron at the active site (22). The structure, determined from crystals of NDO that were reduced with dithionite and then exposed to oxygen, reveals that dioxygen binds side-on close to the mononuclear iron at the active site (Fig. 1B). The distances between the oxygen atoms and iron are 2.2 and 2.3 Å, respectively. The distance between the oxygen atoms was originally fixed at 1.45 Å during refinement (23). The O-O distance, upon crystallographic refinement, converged to 1.4 Å. The refined distance between the oxygen atoms suggests that the structure contains a peroxide species, but the resolution of 1.75 Å does not definitively permit such an assignment. Nor do available spectroscopic studies give a reliable conclusion. Electron paramagnetic resonance (EPR) studies, which were done under different conditions from those of the crystallographic studies, show that a small amount of the mononuclear ferrous iron is oxidized upon exposure to oxygen when oxygen alone is allowed to react with the fully reduced enzyme (17). The reaction with oxygen is much faster with substrate present.

Figure 1

(All panels are stereopairs.) (A) Binding of naphthalene at the active site of NDO. The gray 2F obsF calc map is contoured at 1.0 σ. (B) Binding of dioxygen to the mononuclear iron in the absence of substrate. The gray 2F obsF calc map is contoured at 1.15 σ and the green F obsF calc map (computed before the dioxygen molecule was modeled) at 3.8 × RMS (root mean square). (C) Binding of oxygen to the mononuclear iron in the presence of indole. The gray 2F obsF calcmap is contoured at 1.0 σ and the green F obsF calc map (computed before the dioxygen molecule was modeled) at 4.0 × RMS. (D) Naphthalenecis-dihydrodiol bound to the active site of NDO. The gray 2F obsF calc map is contoured at 1.0 σ. Superposition of the product complex and the substrate complex shows that the positions of the rings in the product and the substrate are similar. The product cannot move any closer to the iron, as it would bring the O from the product into van der Waals short contact with the His ligand of the Fe. The current distance between the O and the N of His is 2.9 Å. Color code: yellow, carbon; blue, nitrogen; red, oxygen; purple, iron.

In a similar experiment, reduced crystals soaked with indole were exposed to oxygen in a pressure cell at –17°C. The structure obtained from these crystals, besides having indole bound, exhibited clear density for a dioxygen species bound side-on with both oxygens coordinated to the iron (Fig. 1C). The turnover reaction is apparently slow under these conditions because no product was visible in the electron density maps. The refined iron-oxygen distances are 1.8 and 2.0 Å and the refined distance between the oxygen atoms converges to about 1.4 Å. The temperature factors of the two oxygen atoms are higher than those of the surrounding atoms (32 and 39; Fe is 20). The bound substrate also has B-factors comparable to the dioxygen species. This indicates that the substrate and dioxygen species have high but not full occupancy. All attempts to model the structure with one bound oxygen (water/hydroxide) resulted in excess residual electron density (23). The structure of the fully oxidized enzyme shows that two water molecules bind differently to the ferric ion in a regular octahedral coordination (fig. S3), so two water molecules can be excluded as an interpretation of the ternary complex.

Both naphthalene and indole are substrates for NDO. A comparison of the binding of naphthalene and indole (22) in the active site of NDO shows that they bind similarly (fig. S4). It is therefore reasonable to suggest that the structure of the ternary complex of enzyme:indole:dioxygen also depicts the binding mode for all substrates that undergo cis-dihydroxylation by NDO. This ternary complex is probably as close to a reactive complex as possible, and its observation might be due to the low temperature at which the experiment was carried out.

The structure determined from NDO crystals in the resting state soaked with naphthalene Cis-dihydrodiol showed this product bound at the active site with both oxygen atoms coordinated to the iron (Fig. 1D). The oxygen-iron distances in this complex are longer than normal (iron-oxygen) coordination distances (2.8 Å).

In the absence of the observation of a side-on–bound dioxygen and solely on the basis of the structure of an indole-oxygen adduct, we previously suggested a sequential mechanism (22). The indole-oxygen adduct (22) left a number of questions unanswered. The structures presented here form a firm basis to propose a concerted mode of attack and provide a better explanation for thecis-specificity of the dihydroxylation reaction.

The series of NDO complex structures with substrate (Fig. 1A; Fig. 2A, 4), oxygen (Fig. 1B; Fig. 2A, 3), substrate and oxygen (Fig. 1C; Fig. 2A, 5), and product (Fig. 1D; Fig. 2A, 6) represent states along a reaction pathway. The arrangement of the oxygen atoms in the ternary complex (Fig. 1C; Fig. 2A, 5) positions the oxygen atoms almost parallel to those observed in the product complex, albeit closer to the iron. Our finding that oxygen binds side-on in both a binary and a ternary complex has several important implications for the cis-dihydroxylation reaction catalyzed by NDO. In a side-on–bound dioxygen species, both oxygen atoms are polarized similarly. This, in addition to the geometric arrangement of the bound dioxygen species in van der Waals contact to the double bond of the substrate, suggests a concerted mechanism where both oxygen atoms react with the carbon atoms of the substrate double bond (Fig. 2B, a). Such a reaction would explain the characteristic cis-stereospecific addition of both oxygen atoms to substrates by NDO.

Figure 2

(A) Scheme showing how the different structures in Fig. 1 can be arranged to follow the dihydroxylation reaction catalyzed by NDO. Naphthalene and indole are both substrates for the enzyme, and we have used them interchangeably in different studies of the enzyme (23). For simplicity we show only naphthalene here. The structures are as follows:1, the resting enzyme with oxidized Rieske center and ferrous active site [Protein Data Bank (PDB) code 1O7H];2, the reduced enzyme (PDB code 1O7W); 3, binary dioxgen complex (PDB code 1O7M); 4, binary substrate complex, structures with both indole and naphthalene [PDB codes 1EG9 (indole) and 1O7G (naphthalene)]; 5, ternary substrate dioxygen species, structure with indole (PDB code 1O7N); and6, product naphthalene cis-1,2-dihydrodiol (PDB code 1O7P). [2Fe-2S] refers to the nearest Rieske iron-sulfur cluster. (B) Chemical steps in the dioxygenation reaction carried out by Rieske dioxygenases.

Further investigation will be needed to determine whether the reactive peroxide is protonated (Fig. 2B, b). If protonated, protons are probably delivered through a water channel connected to Asn201, which is at hydrogen bond distance to the bound dioxygen species. This intermediate could be formed in the reaction of NDO with oxygen or hydrogen peroxide, as proposed by Wolfe and Lipscomb (17, 18).

The unique feature of NDO that sets it apart from P450 is its ability to catalyze stereospecific addition of dioxygen to aromatic compounds, resulting in the formation ofcis-dihydrodiols (12, 24). For cytochrome P450 and MMO, transient high iron oxidation states are required for the necessary oxygen cleavage (25); this is not the case for NDO, where both atoms of dioxygen react in concert with the substrate. The four electrons required for catalysis are one each from the active-site ferrous iron and the Rieske center, and two from the double bond of the substrate. An important difference in the geometry of the active sites of cytochrome P450 and NDO is that the active-site iron is highly accessible in NDO, with a large part of its surface available for the binding of both oxygen atoms to iron. The favorable position of the substrate and Asn201 further favors side-on binding. It is possible that for cytochrome P450 and other heme proteins, the presence of the planar heme group reduces the accessible surface of iron, resulting in an end-on binding of oxygen.

We have directly observed a side-on dioxygen species bound to iron. Computational studies (26) have predicted the existence of this species, and there are spectroscopic data consistent with side-on binding (27, 28). We suggest that side-on binding of oxygen as a starting point for oxygen activation by NDO has direct implications for the mechanism of dihydroxylation of NDO in terms of the stereo- and regiospecific reactions catalyzed by the enzyme (29). The results presented here provide a new basis for further computational and structural investigations of oxygen activation by biological systems and may facilitate the design of chemical catalysts capable of cis-dihydroxylation.

Supporting Online Material

www.sciencemag.org/cgi/content/full/299/5609/1039/DC1

Materials and Methods

References

Figs. S1 to S4

  • * To whom correspondence should be addressed. E-mail: s-ramaswamy{at}uiowa.edu

REFERENCES AND NOTES

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