P460 of hydroxylamine oxidoreductase of Nitrosomonas europaea: Soret resonance Raman evidence for a novel heme-like structure

P460, an iron-containing chromophore at the active site of Hydroxylamine Oxidoreductase of the ammonia-oxidizing bacterium Nitrosomonas europaea, is a macrocycle of unknown structure with a Soret-like 460-nm absorption band in the ferrous form. The pigment can also be isolated in a peptide, "P460-Fragment". Resonance Raman spectroscopy (lambda ex = 457.9 nm) suggests that P460 is a new type of heme with symmetry properties lower than those of protophorphyrin IX or chlorins and similar to those of chlorophylls and isobacteriochlorins. Some of the resonance Raman vibrations of P460 are shifted in HAO as compared to those of P460-Fragment.     

Mutagenesis of hydroxylamine oxidoreductase in Nitrosomonas europaea by transformation and recombination.

Mutagenesis of Nitrosomonas europaea was achieved by electroporation and recombination. To demonstrate this, an aminoglycoside 3'-phosphotransferase (kan) gene was specifically inserted into each of the three gene copies of hao individually. Southern hybridizations and PCR analysis showed the incorporation of the kan gene at the chosen genetic loci. The isolation of mutant strains was achieved in 7 to 14 days when the strains were grown on solid medium. The induced mutations were stable even in the absence of kanamycin-selective pressure for periods of up to 45 days in culture. The mutant strains did not show an observable phenotype different from that of the wild type when grown under the same conditions.     

Quaternary structure of the hydroxylamine oxidoreductase from Nitrosomonas europaea

The hydroxylamine oxidoreductase from Nitrosomonas europaea was prepared to apparent electrophoretic homogeneity. Electron microscopy of negatively stained preparations of the sample revealed an overall diameter of about 8.8 nm of the enzyme particle. The native structure was determined as a tetrahedron-like assembly of identical subunits exhibiting four protein masses.Abbreviations ESI Electron spectroscopic imaging - HAO Hydroxylamine oxidoreductase     

Crystallization and preliminary X-ray studies of hydroxylamine oxidoreductase from Nitrosomonas europaea.

Hydroxylamine oxidoreductase [EC 1.7.3.4] from Nitrosomonas europaea was crystallized by the microdialysis method using ammonium sulfate. Its space group is P63 with cell dimensions of a = b = 96.4 A and c = 266.2 A. Its molecular weight was determined to be 190,000-195,000 by the X-ray small angle scattering and ultracentrifugal methods.     

Highly purified hydroxylamine oxidoreductase derived from Nitrosomonas europaea. Some physicochemical and enzymatic properties.

Hydroxylamine oxidoreductase [EC 1.7.3.4] of Nitrosomonas europaea was purified to an electrophoretically homogeneous state and some of its properties were studied. The molecular weight of the enzyme as determined by gel filtration on Sephadex G150 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate is 175,000-180,000, while the minimum molecular weight per heme determined from the dry weight and heme content is 17,500. The enzyme is a C-type cytochrome; its reduced form shows absorption peaks at 418 (gamma peak), 521 (beta peak), 553 (alpha peak), and 460 nm (due to an unidentified chromophore). Although the alpha peak at 553 nm has a shoulder at 559 nm, the enzyme does not posses protoheme or a cytochrome b subunit. It seems likely that the enzyme molecule possess heme c molecules in different states. The enzyme reacts rapidly with various eukaryotic cytochromes c, but does not react with "bacterial-type" cytochromes c. Although the enzyme does not react with cytochrome c-552 (N. europaea), another C-type cytochrome of the organism, cytochrome c-554 (N. europaea) acts as an electron acceptor for the enzyme.     

The roles of the three gene copies encoding hydroxylamine oxidoreductase in Nitrosomonas europaea

The nitrifying bacterium Nitrosomonas europaea contains three copies of the gene (hao) encoding hydroxylamine oxidoreductase (HAO), the second enzyme in the nitrification pathway which oxidizes NH(2)OH to NO(2)(-). The nucleotide sequences of the hao genes differ by only one nucleotide. Two of the three gene copies have identical promoter sequences, while the third promoter has a different nucleotide sequence. Mutant strains with two of the three copies of hao inactivated were created by insertional inactivation, using DNA cassettes containing kanamycin- and gentamycin-resistance genes. All three double-mutant combinations were obtained. These double mutants were phenotypically identical under the conditions tested. Two of these double mutants were similar to wild-type cells or cells having a single hao copy inactivated regarding growth rates or hydroxylamine-dependent O(2) uptake activity, but had only about 50% of the wild-type level of in vitro HAO activity and hao mRNA. The third hao double mutant had an unstable genotype, resulting in recombination of the gentamycin marker into another copy of hao. The N. europaea genomic sequence was recently completed, revealing the locations of the copies of hao and other nitrification genes. Comparison with the arrangement of hao genes in the closely related strain, Nitrosomonas sp. strain ENI-11, showed a similar organization.     

Spectroscopic and rapid kinetic studies of reduction of cytochrome c554 by hydroxylamine oxidoreductase from Nitrosomonas europaea.

During oxidation of hydroxylamine, hydroxylamine oxidoreductase (HAO) transfers two electrons to tetraheme cytochrome c554 at rates sufficient to account for physiological rates of oxidation of ammonia to nitrite in Nitrosomonas europaea. Spectroscopic changes indicate that the two electrons are taken up by a high-potential pair of hemes (E degrees' = +47 mV) (one apparently high spin and one low spin). During single-turnover experiments, in which the reduction of oxidized cytochrome c554 by NH2OH-reduced HAO is monitored, one electron is taken up by the high-spin heme at a rate too fast to monitor directly (greater than 100 s-1) but which is inferred either by a loss of amplitude (relative to that observed under multiple-turnover conditions) or is slowed down by increasing ionic strength (greater than or equal to 300 mM KCl). The second electron is taken up by the low-spin heme at a 10-30-fold slower rate. The latter kinetics appear multiphasic and may be complicated by a transient oxidation of HAO due to the rapid transfer of the first electron into the high-spin heme of cytochrome c554. Under multiple-turnover conditions, a "slower" rate of reduction is observed for the high-spin heme of cytochrome c554 with a maximum rate constant of approximately 30 s-1, a value also obtained for the reduction, by NH2OH, of the cytochrome c554 high-spin heme within an oxidized HAO/c554 complex. Under these conditions, the maximum rate of reduction of the low-spin heme was approximately 11.0 s-1. Both rates decreased as the concentration of cytochrome c554 was increased above the concentration of HAO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational analysis of the multicopy hao gene coding for hydroxylamine oxidoreductase in Nitrosomonas sp. strain ENI-11

The ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 contains three copies of the hao gene (hao1, hao2, and hao3) coding for hydroxylamine oxidoreductase (HAO). Three single mutants (hao1::kan, hao2::kan, or hao3::kan) had 68 to 75% of the wild-type growth rate and 58 to 89% of the wild-type HAO activity when grown under the same conditions. A double mutant (hao1::kan and hao3::amp) also had 68% of the wild-type growth and 37% of the wild-type HAO activity.     

N2O production from hydroxylamine oxidation and corresponding hydroxylamine oxidoreductase involved in a heterotrophic nitrifier A. faecalis strain NR

Bioprocess and Biosystems Engineering - N2O production from NH2OH oxidation involved in a heterotrophic nitrifier Alcaligenes faecalis strain NR was studied. 15N-labeling experiments showed that...     

Physical map location of the multicopy genes coding for ammonia monooxygenase and hydroxylamine oxidoreductase in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11

Pulsed-field gel electrophoresis of PmeI digests of the Nitrosomonas sp. strain ENI-11 chromosome produced four bands ranging from 1,200 to 480 kb in size. Southern hybridizations suggested that a 487-kb PmeI fragment contained two copies of the amoCAB genes, coding for ammonia monooxygenase (designated amoCAB(1) and amoCAB(2)), and three copies of the hao gene, coding for hydroxylamine oxidoreductase (hao(1), hao(2), and hao(3)). In this DNA fragment, amoCAB(1) and amoCAB(2) were about 390 kb apart, while hao(1), hao(2), and hao(3) were separated by at least about 100 kb from each other. Interestingly, hao(1) and hao(2) were located relatively close to amoCAB(1) and amoCAB(2), respectively. DNA sequence analysis revealed that hao(1) and hao(2) shared 160 identical nucleotides immediately upstream of each translation initiation codon. However, hao(3) showed only 30% nucleotide identity in the 160-bp corresponding region.