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Reduction of nitrous oxide to dinitrogen by a mixed valent tricopper-disulfido cluster.

ABSTRACT: The greenhouse gas N(2)O is converted to N(2) by a mu-sulfido-tetracopper active site in the enzyme nitrous oxide reductase (N(2)OR) via a process postulated to involve mu-1,3 coordination of N(2)O to two Cu(I) ions. In efforts to develop synthetic models of the site with which to test mechanistic hypotheses, we have prepared a localized mixed valent Cu(II)Cu(I)(2) cluster bridged in a mu-eta(2):eta(1):eta(1) fashion by disulfide, [L(3)Cu(3)(mu(3)-S(2))]X(2) (L = 1,4,7-trimethyl-triazacyclononane, X = O(3)SCF(3)(-) or SbF(6)(-)). This cluster exhibits spectroscopic features superficially similar to those of the active site in N(2)OR and reacts with N(2)O to yield N(2) in a reaction that models the function of the enzyme. Computations implicate a transition state structure that features mu-1,1-bridging of N(2)O via its O-atom to a [L(2)Cu(2)(mu-S(2))](+) fragment and provide chemical precedence for an alternative pathway for N(2)O reduction by N(2)OR.

SUBMITTER: Bar-Nahum I

PROVIDER: S-EPMC2880652 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Reduction of nitrous oxide to dinitrogen by a mixed valent tricopper-disulfido cluster.

Bar-Nahum Itsik I Gupta Aalo K AK Huber Stefan M SM Ertem Mehmed Z MZ Cramer Christopher J CJ Tolman William B WB

Journal of the American Chemical Society 20090301 8

The greenhouse gas N(2)O is converted to N(2) by a mu-sulfido-tetracopper active site in the enzyme nitrous oxide reductase (N(2)OR) via a process postulated to involve mu-1,3 coordination of N(2)O to two Cu(I) ions. In efforts to develop synthetic models of the site with which to test mechanistic hypotheses, we have prepared a localized mixed valent Cu(II)Cu(I)(2) cluster bridged in a mu-eta(2):eta(1):eta(1) fashion by disulfide, [L(3)Cu(3)(mu(3)-S(2))]X(2) (L = 1,4,7-trimethyl-triazacyclononan ...[more]

PMID: 19206272

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Project description:One of the major challenges for the bioremediation application of microbial nitrous oxide (N2O) reduction is its oxygen sensitivity. While a few strains were reported capable of reducing N2O under aerobic conditions, the N2O reduction kinetics of phylogenetically diverse N2O reducers are not well understood. Here, we analyzed and compared the kinetics of clade I and clade II N2O-reducing bacteria in the presence or absence of oxygen (O2) by using a whole-cell assay with N2O and O2 microsensors. Among the seven strains tested, N2O reduction of Stutzerimonas stutzeri TR2 and ZoBell was not inhibited by oxygen (i.e., oxygen tolerant). Paracoccus denitrificans, Azospirillum brasilense, and Gemmatimonas aurantiaca reduced N2O in the presence of O2 but slower than in the absence of O2 (i.e., oxygen sensitive). N2O reduction of Pseudomonas aeruginosa and Dechloromonas aromatica did not occur when O2 was present (i.e., oxygen intolerant). Amino acid sequences and predicted structures of NosZ were highly similar among these strains, whereas oxygen-tolerant N2O reducers had higher oxygen consumption rates. The results suggest that the mechanism of O2 tolerance is not directly related to NosZ structure but is rather related to the scavenging of O2 in the cells and/or accessory proteins encoded by the nos cluster. IMPORTANCE Some bacteria can reduce N2O in the presence of O2, whereas others cannot. It is unclear whether this trait of aerobic N2O reduction is related to the phylogeny and structure of N2O reductase. The understanding of aerobic N2O reduction is critical for guiding emission control, due to the common concurrence of N2O and O2 in natural and engineered systems. This study provided the N2O reduction kinetics of various bacteria under aerobic and anaerobic conditions and classified the bacteria into oxygen-tolerant, -sensitive, and -intolerant N2O reducers. Oxygen-tolerant N2O reducers rapidly consumed O2, which could help maintain the low O2 concentration in the cells and keep their N2O reductase active. These findings are important and useful when selecting N2O reducers for bioremediation applications.

Project description:N2O-reducing organisms with nitrous oxide reductases (NosZ) are known as the only biological sink of N2O in the environment. Among the most abundant nosZ genes found in the environment are nosZ genes affiliated with the understudied Gemmatimonadetes phylum. In this study, a unique regulatory mechanism of N2O reduction in Gemmatimonas aurantiaca strain T-27, an isolate affiliated with the Gemmatimonadetes phylum, was examined. Strain T-27 was incubated with N2O and/or O2 as the electron acceptor. Significant N2O reduction was observed only when O2 was initially present. When batch cultures of strain T-27 were amended with O2 and N2O, N2O reduction commenced after O2 was depleted. In a long-term incubation with the addition of N2O upon depletion, the N2O reduction rate decreased over time and came to an eventual stop. Spiking of the culture with O2 resulted in the resuscitation of N2O reduction activity, supporting the hypothesis that N2O reduction by strain T-27 required the transient presence of O2 The highest level of nosZ transcription (8.97 nosZ transcripts/recA transcript) was observed immediately after O2 depletion, and transcription decreased ∼25-fold within 85 h, supporting the observed phenotype. The observed difference between responses of strain T-27 cultures amended with and without N2O to O2 starvation suggested that N2O helped sustain the viability of strain T-27 during temporary anoxia, although N2O reduction was not coupled to growth. The findings in this study suggest that obligate aerobic microorganisms with nosZ genes may utilize N2O as a temporary surrogate for O2 to survive periodic anoxia.IMPORTANCE Emission of N2O, a potent greenhouse gas and ozone depletion agent, from the soil environment is largely determined by microbial sources and sinks. N2O reduction by organisms with N2O reductases (NosZ) is the only known biological sink of N2O at environmentally relevant concentrations (up to ∼1,000 parts per million by volume [ppmv]). Although a large fraction of nosZ genes recovered from soil is affiliated with nosZ found in the genomes of the obligate aerobic phylum Gemmatimonadetes, N2O reduction has not yet been confirmed in any of these organisms. This study demonstrates that N2O is reduced by an obligate aerobic bacterium, Gemmatimonas aurantiaca strain T-27, and suggests a novel regulation mechanism for N2O reduction in this organism, which may also be applicable to other obligate aerobic organisms possessing nosZ genes. We expect that these findings will significantly advance the understanding of N2O dynamics in environments with frequent transitions between oxic and anoxic conditions.

Project description:Aerobic methanotrophic activity is highly dependent on copper availability, and methanotrophs have developed multiple strategies to collect copper. Specifically, when copper is limiting (ambient concentrations less than 1 μM), some methanotrophs produce and secret a small modified peptide (less than 1,300 Da) termed methanobactin (MB) that binds copper with high affinity. As MB is secreted into the environment, other microbes that require copper for their metabolism may be inhibited as MB may make copper unavailable; e.g., inhibition of denitrifiers as complete conversion nitrate to dinitrogen involves multiple enzymes, some of which are copper-dependent. Of key concern is inhibition of the copper-dependent nitrous oxide reductase (NosZ), the only known enzyme capable of converting nitrous oxide (N2O) to dinitrogen. Herein, we show that different forms of MB differentially affect copper uptake and N2O reduction by Pseudomonas stutzeri strain DCP-Ps1 (that expresses clade I NosZ) and Dechloromonas aromatica strain RCB (that expresses clade II NosZ). Specifically, in the presence of MB from Methylocystis sp. strain SB2 (SB2-MB), copper uptake and nosZ expression were more significantly reduced than in the presence of MB from Methylosinus trichosporium OB3b (OB3b-MB). Further, N2O accumulation increased more significantly for both P. stutzeri strain DCP-Ps1 and D. aromatica strain RCB in the presence of SB2-MB versus OB3b-MB. These data illustrate that copper competition between methanotrophs and denitrifying bacteria can be significant and that the extent of such competition is dependent on the form of MB that methanotrophs produce. IMPORTANCE Herein, it was demonstrated that the different forms of methanobactin differentially enhance N2O emissions from Pseudomonas stutzeri strain DCP-Ps1 (harboring clade I nitrous oxide reductase) and Dechloromonas aromatica strain RCB (harboring clade II nitrous oxide reductase). This work contributes to our understanding of how aerobic methanotrophs compete with denitrifiers for the copper uptake and also suggests how MBs prevent copper collection by denitrifiers, thus downregulating expression of nitrous oxide reductase. This study provides critical information for enhanced understanding of microbe-microbe interactions that are important for the development of better predictive models of net greenhouse gas emissions (i.e., methane and nitrous oxide) that are significantly controlled by microbial activity.

Dataset Information

Reduction of nitrous oxide to dinitrogen by a mixed valent tricopper-disulfido cluster.

Publications

Reduction of nitrous oxide to dinitrogen by a mixed valent tricopper-disulfido cluster.

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