Nitrite reduction by nitrite reductases and related model systems, and the role of linkage isomerism therein

Radu Silaghi-Dumitrescu

Nitrite reduction by nitrite reductases and related model systems, and the role of linkage isomerism

Reduction of nitrite to nitric oxide is essential in certain living species, and has in fact recently been proposed to be an important secondary function of hemoglobin in humans (cf Gladwin, NATURE - CHEM BIOL 2005, 245). In vivo, nitrite reduction is accomplished by metalloenzymes, involving direct metal-nitrite coordination. Recently, we proposed that linkage nitro/nitrito isomerism is an essential part of the mechanism in copper and in heme d1-containing nitrite reductases (cf Silaghi, INORG CHEM 2004, 256). Recent experimental work has also shown involvement of linkage isomerism in catalytic reduction of nitrite by free hemes and related small complexes in solution.

Under anaerobic conditions, many microorganisms can sustain growth by using nitrate as respiratory terminal electron acceptor. Much like dioxygen, partially reduced products of nitrate may be toxic to living cells. While respiratory dioxygen reduction delivers all four electrons in one single step (O₂ + 4H⁺ +4e^- -> 2H₂O), respiratory nitrate reduction in bacteria and archaea is more complex, proceeding stepwise on more than one possible pathway. Bacteria in fact exhibit a wide range of metabolic reactions with various oxides of nitrogen, sometimes referred to as the bacterial nitrogen cycle, which is diagrammed in Figure 1. [Richardson et al, Curr. Opin. Chem. Biol. 1999, 207]

Figure 1. The bacterial nitrogen cycle. The oxidation states of nitrogen are shown in parentheses for each species.

Nitrate and nitrite may be reduced to ammonia for the purpose of nitrogen assimilation (incorporation into organic matter, non-energy conserving) or dissimilation (using nitrate as respiratory electron acceptor, i.e., energy-conserving, but without incorporating the final reduced product into organic matter). Reduction of dinitrogen to ammonia is termed nitrogen fixation. All the known enzymes catalyzing the reactions of the nitrogen cycle are metalloenzymes. Nitrate reduction to nitrite is catalyzed by nitrate reductases, which are molybdopterin enzymes. The subsequent reduction of nitrite is catalyzed by two types of nitrite reductases: those reducing nitrite to ammonia (cytochrome c nitrite reductase, siroheme-containing nitrite reductase) and those reducing nitrite to nitric oxide (copper-containing nitrite reductase, cytochrome cd1 nitrite reductase) [Einsle et al, JACS 2002, 11737; Silaghi INORG CHEM 2004, 256; Silaghi EUR J INORG CHEM 2003, 1048]. For those nitrite reductases catalyzing nitrite reduction to ammonia, NO and hydroxylamine are proposed to constitute reaction intermediates, which are never released from the active site. In cytochrome c nitrite reductase, NO is reduced to NH₄⁺, in a mechanism proposed to involve an {FeNO}⁶→{FeNO}⁷→{FeNO}⁸ sequence (cf Enemark-Feltham notation) at the lysine-ligated heme active site [Einsle et al, JACS 2002, 11737; Silaghi EUR J INORG CHEM 2003]. On the other hand, cytochrome cd1 nitrite reductase, which reduces nitrite to NO in vivo, can also reduce NO to N₂O, but only in vitro [Silaghi INORG CHEM 2004]. When produced by an NO-forming nitrite reductase, nitric oxide is further reduced to N₂O by nitric oxide reductases, which, contain either cytochrome bd-or P450-type active sites [Silaghi EUR J INORG CHEM 2003]. Nitrous oxide (a relatively inert gas) may either be released as endproduct or further reduced to molecular nitrogen by nitrous oxide reductases (which are multinuclear copper proteins). Molecular nitrogen is reduced to ammonia by nitrogenases.

Thus, biological nitrate reduction leads to one of three end-products: ammonia (which may be readily incorporated into organic matter), nitrous oxide, or dinitrogen. Additionally, ANAMOX, a little understood biological process is proposed to generate molecular nitrogen from nitrate and ammonia via hydroxylamine and hydrazine.

Nitrification is an oxidative pathway starting from ammonia and also generates partially reduced nitrogen oxides. Thus, oxidation of ammonia to hydroxylamine is catalyzed by ammonia monooxygenase (a little understood enzyme, apparently related to the particulate methane monooxygenase), and is followed by oxidation of hydroxylamine to nitrite by hydroxylamine oxidase (a multiheme enzyme). These oxidations provide electrons necessary for respiratory electron transfer chains that facilitate lithotrophic (“rock-eating”, i.e. extracting energy from inorganic compounds) growth. [Richardson et al, Curr. Opin. Chem. Biol. 1999, 207]

In addition to the above described nitrogen cycle, reduction of nitrite to nitric oxide has also recently been proposed to be an important secondary function of hemoglobin in humans, whereby the vasodilator molecule nitric oxide (of Nobel Prize fame) would be generated [Gladwin, NATURE CHEM BIOL 2005, 245]. In fact, precisely due to this nitrite reductase chemistry, nitrite is currently being introduced in clinical trials for cardiovascular diseases in the USA. The mechanisms whereby nitrite is reduced by hemes thus begin to entail increasing medical relevance.

We have recently proposed that linkage nitro/nitrito isomerism is an essential part of the mechanism in the cases of copper and of heme d1-containing nitrite reductases (NIR) [Silaghi INORG CHEM 2004, 256]. Nitrite reduction by cytochrome cd₁ nitrite reductase (cd₁NIR) has long been proposed to occur (cf. Scheme 1) via N-coordination of nitrite to the d₁ heme of cd₁NIR. Protonation of a nitrite oxygen atom within the ferrous-nitrite complex would lead to release of a water molecule, forming a weakly-boundcomplex, that subsequently decays via NO liberation. Nitrite and nitric oxide adducts of the d₁ heme in cd₁NIR have been characterized experimentally and computationally [Williams et al, NATURE 1997, 406; Silaghi INORG CHEM 2004, 256; Richter et al J BIOL CHEM 2002, 3093].

Scheme 1

Our group has however explored an alternative possibility, involving linkage isomerism of the nitrite at the NIR site. [Silaghi INORG CHEM 2004; Silaghi REV ROUM CHIM 2004, 496] Density functional theory results were thus reported on the previously unexplored O-binding of nitrite to ferrous and ferric cd1NIR. Although the N- isomer (nitro) is energetically favored over the O-nitrite (nitrito), even one single strong hydrogen bond may provide the energy required to put the two isomers on level terms. When hydrogen bonding existent at the cd1NIR active site was accounted for in the computational model, the O-nitrite isomer is found to spontaneously protonate and thus yield a ferric-hydroxo species, liberating nitric oxide. An O-nitrite ferrous cd1NIR complex appears to be an energetically-feasible intermediate for nitrite reduction. O-coordination would offer an advantage since the end-product of nitrite reduction would be a ferric-hydroxo/water complex, rather than the more kinetically inert iron-nitrosyl complex implied by the N-nitrite mechanism. Some of this computational data is illustrated in Figure 2.

Figure 2. Optimized geometries for O- and N- ferrous-nitrite models of the cd₁NIR active site. Distances for the corresponding ferric-nitrite models are given in parentheses [Silaghi INORG CHEM 2004].

Our revised catalytic cycle for cd1NIR is thus illustrated in Scheme2. This mechanism, unlike the one in Scheme 1, reconciles for the first time the cd1NIR chemistry with the puzzling fact that Fe(III)-NO is kinetically inert and hence cannot possibly be a part of the cd1NIR catalytic cycle [Silaghi INORG CHEM 2004].

Scheme 2

The same process as in cd1NIR, i.e. reduction of nitrite to nitric oxide, is also catalyzed by copper-containing NIR (Cu-NIR) [Silaghi, J INORG BIOCHEM 2006, 396; Tocheva et al, SCIENCE 2004, 867]. The proposed catalytic mechanism, illustrated in Scheme 3, has recently been confirmed by our own computational investigations. However, we have found that even for Cu-NIR, nitrite linkage isomerism is an intrinsic property of the metal site, a property which the protein has to modulate in order to achieve its goal of rapid catalytic turnover. Figure 4 illustrates some of our computational results leading to these conclusions [Silaghi, J INORG BIOCHEM 2006, 396].

Scheme 3

Figure 4. Overlay (viewed along the and perpendicular to the approximate Cu-nitrite axis, respectively) of the experimental (pdb code 1SJM) and calculated structures of the Cu(II)-nitrite adducts. Color coding: iron, green; carbon, grey; nitrogen, blue; oxygen, red; hydrogen, white.

Independent experimental work has also recently supported the concept that linkage isomerism has a profound influence on the products and mechanisms of catalytic reduction of nitrite by free hemes and related small complexes in solution [Kudrik et al, INORG CHEM 2005, 6470]. In many abiological processes, catalysis involves redox chemistry and takes place at metal centers as in the biological processes. The porphyrin-like molecules and their metallocomplexes play an unique role in these processes. The huge opportunities for modification of the porphyrin core, the strong ability of metalloporphyrinates to coordinate extra-ligands including small molecules give scientists the great possibilities to influence the catalytic process. The additional circumstance that widen a set of possible reaction pathways is the availability of two potential coordination sites in some N,O-containing species. Recent studies [Kudrik et al, INORG CHEM 2003, 618] showed that reactions of nitrate and nitrite with sodium dithionite in the presence of Co^IItetrasulfophthalocyaninate in aqueous alkaline solution lead to different products (nitrous oxide and ammonia, respectively). These striking differences were explained in terms of different structures of the intermediate complex between Co^Iphthalocyaninate and substrate, in which nitrite and nitrate were suggested to coordinate via nitrogen and oxygen, respectively. O-coordination of nitrite had also been proved for ruthenium and manganese porphyrinates [Kudrik et al, INORG CHEM 2005, 6470; Silaghi INORG CHEM 2004].