Effect of Salinity on Hydroxylamine Oxidation in a Marine Ammonia-Oxidizing Gammaproteobacterium, Nitrosococcus oceani strain NS58: Molecular and Catalytic Properties of Tetraheme Cytochrome c-554

Abstract

Tetraheme cytochrome c-554 is a physiological electron acceptor of hydroxylamine oxidoreductase (HAO), a core enzyme of ammonia oxidation in chemoautotrophic nitrifiers. Here we report the purification of cytochrome c-554 from Nitrosococcus oceani strain NS58, a marine gammaproteobacterial ammonia-oxidizing bacterium. The NS58 cytochrome is a 25 kDa-protein having four hemes c. The absorption spectrum of the cytochrome showed peaks at 420 nm, 523 nm, and 554 nm, with shoulders at around 430 nm and 580 nm in the reduced state. In contrast to the highly basic counterpart from the betaproteobacterium Nitrosomonas europaea, the NS58 cytochrome c-554 was an acidic protein whose isoelectric point was 4.6. HAO was also purified, and the reaction with the NS58 cytochrome was found to be salt-tolerant. Compared with the activity observed in a non-salt solution, 60% of the activity remained in a saline concentration comparable to that of seawater.

Isotopomeric characterization of nitrous oxide produced by reaction of enzymes extracted from nitrifying and denitrifying bacteria

“…Nitrosococcus oceani strain NS58 was kindly supplied by Dr. H. Urakawa (Florida Gulf Coast University). Phylogenetic and morphological analyses indicated a close systematic relationship of the bacterium with N. oceani ATCC19707 (Hozuki et al, 2010). The bacterium was cultivated in the (NH 4 ) 2 SO 4 -supplemented artificial seawater (37.8 mM NH + 4 ), of which the pH was buffered to 7.8 by 50 mM MOPS (3-morpholinopropanesulfonic acid) as described in detail in a previous report (Hozuki et al, 2010).…”

“…HAO was purified from the cultivated NS58 cells through three preparative steps, including (NH 4 ) 2 SO 4 fractionation, gel-filtration and hydrogen-bonding chromatography according to a previous report (Hozuki et al, 2010). Catalytic activity of HAO was analyzed by spectrophotometrical measurement of the NH 2 OH-dependent reduction of potassium ferricyanide as reported previously (Hozuki et al, 2010). Briefly, the purified enzyme was mixed with 1 mL of the reaction solution containing 0.1 M sodium phosphate buffer (pH 7.8), 20 µM NH 2 OH and 100 µM potassium ferricyanide.…”

Expression, and Molecular and Enzymatic Characterization of Cu-Containing Nitrite Reductase from a Marine Ammonia-Oxidizing Gammaproteobacterium, <i>Nitrosococcus oceani</i>

“…According to a previous report on the purification of N. europaea nitrite reductase, the enzyme was co-isolated with hydroxylamine oxidoreductase by gel filtration because it is soluble and has a high molecular weight (8, 13); however, in the case of N. oceani NS58, no nitrite reducing activity was detected in any fraction obtained by gel filtration. On the other hand, generation of N 2 O from intact cells of N. oceani by the nitrifier denitrification pathway has been demonstrated by inhibition analysis (5).…”

“…Medium composition and protocol for large-scale cultivation in 10 L volume three times with N. oceani NS58 followed a previous report (13). Genomic DNA of the NS58 was prepared by a standard method.…”

“…Recently, we have been studying the biochemistry of ammonia oxidation and its relative processes in a marine γ-AOB, strain NS58, which was isolated in Tokyo Bay and is phylogenetically very close to N. oceani ATCC19707 (13). In this study, the Cu-containing nitrite reductase of N. oceani NS58 was prepared as a recombinant protein, and its molecular and catalytic properties were analyzed.…”