Hydroxylamine oxidoreductase

The oxidation of hydroxylamine by the ammonia-oxidizing bacteria is catalyzed by hydroxylamine oxidoreductase (Yamanaka and Sakano, 1980). The molecular 

Besides heme P-460 of hydroxylamine oxidoreductase, cytochrome P-460 has been isolated from N. europaea (Erickson and Hooper, 1972 Miller et al., 1984 Numata et al., 1990). Cytochrome P-460 shows a weak activity of hydroxylamine oxidoreductase, and its molecule is composed of 3 subunits of 18 kDa. As the amino acid sequence of the cytochrome differs from that of hydroxylamine oxidoreductase and DNA encoding the cytochrome is found to be different from that of the oxidoreductase, the cytochrome is not a proteolytic fragment of the oxidoreductase (Bergman and Hooper, 1994b). Indeed, cytochrome P-460 has recently been crystallized and its spatial structure has been determined (Pearson et al., 2007). Its heme is a modified heme C in which lysine residue links to 

A question occurs as to why the bacterial enzyme has such a complicated structure, because hydroxylamine is oxidized to nitrite by the catalysis of ferric ion under aerobic conditions. In the nonenzymatic reaction, molecular oxygen is incorporated into nitrite formed by the oxidation of hydroxylamine, while the oxygen atom of water is incorporated into nitrite formed by the enzymatic oxidation of hydroxylamine (see below) (Yamanaka and Sakano, 1980 Andersson and Hooper, 1983). The mechanism in the bacterial oxidation of hydroxylamine will have been devised to reserve efficiently the energy of the reaction for the biosynthesis of adenosine triphosphate (ATP). 

Hydroxylamine oxidoreductase was first purified by Hooper and Nason in 1965. They found that the enzyme catalyzed the reduction of horse ferricytochrome c with hydroxylamine but they found little nitrous acid formed as the result of the reaction nitrite formed was approximately 5% as much as cytochrome c reduced in 0.1 M glycine-NaOH buffer, pH 9.8. As the ammonia-oxidizing bacteria oxidize hydroxylamine to nitrite, they thought that one or more enzymes in addition to the oxidoreductase might participate in the oxidation of hydroxylamine to nitrite or additional factor(s) might be necessary for changing hydroxylamine to nitrite (Hooper et al., 1977). 

In 1980, a student for whom the author was a supervisor at Osaka University found that if the amount of ferricytochrome c used was 10 times as much as that of hydroxylamine added and the reactions were performed in 0.1 M phosphate buffer, pH 8.0, the enzyme catalyzed almost completely the oxidation of hydroxylamine to nitrite (Yamanaka and Sakano, 1980). Thus, it has been established that hydroxylamine is oxidized to nitrite by the catalysis of hydroxylamine oxidoreductase itself. Furthermore, the formation of nitrite by the enzymatic oxidation of hydroxylamine has been found to occur even under anaerobic conditions if a sufficient amount of the electron acceptor is present. Therefore, the enzymatic oxidation of hydroxylamine itself does not require molecular oxygen, though ferrocytochrome c-554 [native ferrocytochrome c] formed by the dehydrogenation of hydroxylamine has to be eventually oxidized by atmospheric oxygen, in vivo. 

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Hydroxylamine oxidoreductase (HAO) (bacteria) c-type cytochrome heme-containing enzyme. Oxidizes HA to nitrite (N02 ) 8, 33, 47... 

Figure 18-19 The ammonia oxidation system of the bacterium Nitrosomonas. Oxidation of ammonium ion (as free NH3) according to Eq. 18-17 is catalyzed hy two enzymes. The location of ammonia monooxygenase (step a) is uncertain but hydroxylamine oxidoreductase (step b) is periplas-mic. The membrane components resemble complexes I, III, and IV of the mitochondrial respiratory chain (Fig. 18-5) and are assumed to have similar proton pumps. Solid green lines trace the flow of electrons in the energy-producing reactions. This includes flow of electrons to the ammonia monoxygenase. Complexes HI and IV pump protons out but complex I catalyzes reverse electron transport for a fraction of the electrons from hydroxylamine oxidoreductase to NAD+. Modified from Blaut and Gottschalk.315...

N. Igarashi and N. Tanaka, Hydroxylamine Oxidoreductase, in Handbook of Metalloproteins , eds. A. Messerschmidt, R. Huber, T. Poulos, and K. Wieghardt, John Wiley Sons New York, 2001, p. 454. 

The immediate product of AMO is hydroxylamine, which is further oxidized by hydroxylamine oxidoreductase (HAO) to N02 (Eq. (5.2)). AOA apparently do not possess the hydroxylamine reductase gene, so the pathway of ammonia oxidation in these organisms must be quite different from that outlined here for AOB. Oxygen is also consumed by the terminal oxidase (Eq. (5.3)), as a result of electron transport generating ATP for cellular metabolism. 

Hooper, A. B., and Terry, K. R. (1979). Hydroxylamine oxidoreductase of Nitrosomonas production of nitric-oxide from hydroxylamine. Biochimica et Biophyska Acta 571, 12-20. 

Figure 12 A diagram of the nitrogen cycle with catalyzing enzymes and metal requirements of each step. NIT, nitrogenase AMO, ammonium mono-oxygenase HAO, hydroxylamine oxidoreductase NAR, membrane-bound respiratory nitrate reductase NAP, periplasmic respiratory nitrate reductase NR, assimila-tory nitrate reductase NIR, respiratory nitrite reductase NiR, assimilatory nitrite reductase NOR, nitric oxide reductase N2OR, nitrous oxide reductase.

The enzyme hydroxylamine oxidoreductase catalyzes the oxidation of hydroxylamine to nitrite. This enzyme has long been known to be rich in cytochromes, and it has now been characterized as an complex of three monoheme c-type cytochromes (molecular weight 11000 each)... 

The 24-heme containing hydroxylamine oxidoreductase recently has been reported to contain a diheme cluster in the active site (182). This new cluster might contain a jU,-oxo or a /a-hydroxo bridge, but it is more likely that a histidine is the bridging ligand. 

The ammonia-oxidizing bacteria oxidize ammonia to nitrous acid via hydroxyl-amine (NH2OH) (Lees, 1952 Hofman and Lees, 1953) ammonia is first oxidized to hydroxylamine by the catalysis of ammonia monooxygenase (AMO) (Dua et al., 1979 Hollocher et al., 1981). In this reaction, molecular oxygen is utilized. Then, hydroxylamine formed is oxidized to nitrous acid by the catalysis of hydroxylamine oxidoreductase (HAO). 

In retrospect, it is recognized by the detailed analyses that when horse ferricytochrome c is used as the electron acceptor, nitrite of approximately one fourth as much as horse cytochrome c reduced is usually formed in the oxidation of hydroxylamine catalyzed by hydroxylamine oxidoreductase regardless of the amount of the ferricytochrome c added, if the reactions are performed in 0.1 M phosphate buffer, pH 8.0. As the rate of the reaction (3.4) is not so much slower than that of the reaction (3.3) as assumed previously, the reaction (3.4) seems to occur at a comparable rate to the reaction (3.3). 

An appreciable amount of nitrite is formed, therefore, by the catalysis of hydroxylamine oxidoreductase even in the presence of less ferricytochrome c than its amount four times as much as hydroxylamine. However, when ferricya-nide is used as the electron acceptor for the oxidoreductase, the first step of the reaction in the dehydrogenation of hydroxylamine seems to be much faster than the second step the amount of nitrite enzymatically formed in the case where the ratio of hydroxylamine to ferricyanide is 100 pM to 100 pM is much less than that formed in the case where the ratio is 10 pM to 100 pM. Incidentally, in the enzymatic oxidation of hydroxylamine in 0.1 M glycine-NaOH buffer, pH 9.8, nitrite formed is approximately one third as much as the compound formed in the reactions performed in 0.1 M phosphate buffer, pH 8.0. Glycine may react with NOH to form e.g. N2, and the amount of nitrite formed is diminished. 

In the enzymatic oxidation of hydroxylamine catalyzed by hydroxylamine oxidoreductase, the electron acceptor for the oxidoreductase, cytochrome c-554, should be kept in the oxidized form as much as possible to accept electrons rapidly from hydroxylamine and NOH. For this purpose, sufficient air should be supplied for the bacteria to oxidize ammonia efficiently. If the air supply is not enough to oxidize hydroxylamine to nitrite, nitrous oxide (N20) occurs during the bacterial oxidation of ammonia (Poth, 1986 Anderson et al., 1993). Probably... 

As hydroxylamine oxidoreductase has been described above, some properties of other components involved in the above electron transfer system will be described below. 

Cytochrome c-552 is a monoheme cytochrome c with molecular mass of 9.3 kDa. The structure of the cytochrome has been supposed to be similar to that of Pseudomonas aeruginosa cytochrome c-551 on the basis its reactivity with several oxi-doreductase (Yamanaka and Shinra, 1974). Indeed, its amino acid sequence is very similar to that of P. aeruginosa cytochrome c-551 (Fujiwara et al., 1995 Timkovich et al., 1998). Although cytochrome c-552 does not act as the electron acceptor for hydroxylamine oxidoreductase, it is reduced with hydroxylamine by the catalysis of the enzyme in the presence of a catalytic amount of cytochrome c-554. Furthermore, ferrocytochrome c-552 is oxidized with molecular oxygen by the catalysis of cytochrome c oxidase (cytochrome aa3) as described below (Yamazaki et al., 1985). 

From several heterotrophic nitrifiers, enzymes are obtained which catalyze the reduction of ferricytochrome c in the presence of hydroxylamine like hydroxylamine oxidoreductase of N. europaea does, but they do not have heme C, unlike the N. europaea enzyme (Kurokawa et al., 1985 Wehrfritz et al., 1993, 1997). Hydroxylamine oxidoreductase purified from Alcaligenes faecalis strain TUD has non-heme iron and catalyzes the reduction of ferricyanide with hydroxylamine, but does not catalyze the reduction of ferricytochrome c with hydroxylamine (Otte et al., 1999). 

Arciero DM, Balny C, Hooper AB (1991) Spectroscopic and rapid kinetic studies of reduction of cytochrome c-554 by hydroxylamine oxidoreductase from Nitrosomonas europaea. Biochemistry 30 11466-11472... 

Arciero DM, Hooper AB, Cai M, Timkovich R (1993) Evidence for structure of active site heme P-460 in hydroxylamine oxidoreductase of Nitrosomonas. Biochemistry 32 9370-9378... 

Collinet M-N, Morin D (1990) Characterization of arsenopyrite oxidizing Thiobacillus. Tolerance to arsenite, arsenate and ferric iron. Antonie van Eeeuwenhoek 57 237-244 Collins MJ, Arciero DM, Hooper AB (1993) Optical spectropotentiometric resolution of the hemes of hydroxylamine oxidoreductase. Heme quantitation and pH dependence of Em. J Biol Chem 268 14655-14662... 

Hooper AB, Terry KR (1979) Hydroxylamine oxidoreductase of Nitrosomonas. Production of nitric oxide from hydroxylamine. Biochim Biophys Acta 571 12-20 Hooper AB, Terry KR, Maxwell PC (1977) Hydroxylamine oxidoreductase of Nitrosomonas. Oxidation of diethyldithiocarbamate concomitant with stimulation of nitrite synthesis. Biochim Biophys Acta 462 141-152... 

Iwata S, Ostermeier C, Ludwig B, Michel H (1995) Structure at 2.8A resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature 376 660-669 Jannasch HW, Nelson DC, Wirsen CO (1989) Massive natural occurrence of unusually large bacteria (Beggiatoa sp.) at a hydrothermal deep-sea vent site. Nature 342 834—836 Jetten MSM, de Bruijn P, Kuenen JG (1997) Hydroxylamine metabolism in Pseudomonas PB16 involvement of a novel hydroxylamine oxidoreductase. Antonie van Leeuwenhoek. 71 69-74... 

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