Nitric Oxide Reductase from <i>Paracoccus denitrificans</i> Contains an Oxo-Bridged Heme/Non-Heme Diiron Center

Moënne‐Loccoz, Pierre; Richter, O.-M. H.; Huang, Huaxiong; Wasser, Ian M.; Ghiladi, Reza A.; Karlin, Kenneth D.; Vries, Simon de

doi:10.1021/ja0016295

Cited by 90 publications

(112 citation statements)

References 25 publications

(19 reference statements)

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“…Upon addition of Fe 2þ , however, a decrease of the heme-Fe(III) EPR signals was observed, indicating that the Fe 2þ , when bound to the Fe B site and oxidized by Cu(II)-azurin, is spin-coupled to heme-Fe(III). Such a spin coupling mimics that in NOR (35,(50)(51)(52)(53), suggesting that I107E Fe B Mb models NOR closely, at least in this respect.…”

Section: Resultsmentioning

confidence: 62%

Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin

Lin

Yeung²,

Gao³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

107

140

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A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative Fe B site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by ∼110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu has a different role in redox tuning. More importantly, introducing the second Glu resulted in a ∼100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the Fe B site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more welldefined system to address important chemical and biological issues.biomimetic models | heme-copper oxidase | metalloprotein | protein design | protein engineering R ational design of proteins that mimic both structure and function of more complex native enzymes has been a long soughtafter goal, as the process is an ultimate test of our knowledge and an excellent means to develop advanced biocatalysts (1-3). Although designed proteins that model the structure of native enzymes have been known for a while (4-10), successful designs of proteins that mimic both the structure and function of native enzymes have been reported only recently (11-16). While being able to design such functional proteins is laudable, the impact of such an achievement would be greater if the designed proteins can be used to address fundamental issues in chemistry and biology that are difficult to tackle by other methods. One primary example is the roles of conserved glutamates and metal ions in bacterial nitric oxide reductase (NOR) (17)(18)(19).NO is critical for all life (20). Bacterial denitrification is a crucial part of the nitrogen cycle in nature that involves a four-step, five-electron reduction of nitrate (NO 3 − ) to dinitrogen (N 2 ) (17, 19). Bacterial NOR is a membrane-bound protein that catalyzes one step of this process, namely, the two-electron reduction of NO to N 2 O (17, 19). With no crystal or solution structure available for bacterial NOR to date, sequence alignments and homology modeling (21, 22) have indicated that NOR is structurally homologous to the largest subunit (subunit I) of hemecopper oxidases (HCOs) (23), enzymes that catalyze reduction of O 2 to water. The active sites of both NOR and HCO contain a proximal histidine-coo...

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Section: Resultsmentioning

confidence: 62%

Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin

Lin

Yeung²,

Gao³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

107

140

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“…11 In the oxidized enzyme, the presence of a μ-oxo-group bridging the two iron(III) centers was revealed by isotopic exchange with 18 O-labeled water. 75 77 provided the first direct evidence of a diiron cluster with an Fe-Fe distance ≤ 3.5 Å (Fig. 3).…”

Section: Spectroscopic Characterization Of Denitrifying No Reductasesmentioning

confidence: 93%

Spectroscopic characterization of heme iron–nitrosyl species and their role in NO reductase mechanisms in diiron proteins

Moënne‐Loccoz

2007

Nat. Prod. Rep.

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121

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Nitric oxide (NO) plays an important role in cell signalling and in the mammalian immune response to infection. On its own, NO is a relatively inert radical, and when it is used as a signalling molecule, its concentration remains within the picomolar range. However, at infection sites, the NO concentration can reach the micromolar range, and reactions with other radical species and transition metals lead to a broad toxicity. Under aerobic conditions, microorganisms cope with this nitrosative stress by oxidizing NO to nitrate (NO 3 − ). Microbial hemoglobins play an essential role in this NO-detoxifying process. Under anaerobic conditions, detoxification occurs via a 2-electron reduction of two NO molecules to N 2 O. In many bacteria and archaea, this NOreductase reaction is catalyzed by diiron proteins. Despite the importance of this reaction in providing microorganisms with a resistance to the mammalian immune response, its mechanism remains ill-defined. Because NO is an obligatory intermediate of the denitrification pathway, respiratory NO reductases also provide resistance to toxic concentrations of NO. This family of enzymes is the focus of this review. Respiratory NO reductases are integral membrane protein complexes that contain a norB subunit evolutionarily related to subunit I of cytochrome c oxidase (CcO). NorB anchors one high-spin heme b 3 and one non-heme iron known as Fe B , i.e., analogous to Cu B in CcO. A second group of diiron proteins with NO-reductase activity is comprised of the large family of soluble flavoprotein A found in strict and facultative anaerobic bacteria and archaea. These soluble detoxifying NO reductases contain a non-heme diiron cluster with a Fe-Fe distance of 3.4 Å and are only briefly mentioned here as a promising field of research. This article describes possible mechanisms of NO reduction to N 2 O in denitrifying NO-reductase (NOR) proteins and critically reviews recent experimental results. Relevant theoretical model calculations and spectroscopic studies of the NO-reductase reaction in heme/copper terminal oxidases are also overviewed.

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“…It has been reported that in oxidized Nor, heme b 3 is not coordinated to the protein by its proximal histidine residue (6). In addition, the single oxygen isotope-sensitive ligand vibration observed at 811 cm Ϫ1 in the RR spectrum was attributed to the as (Fe-OFe) of the heme b 3 /non-heme diiron center (7). However, the structural implications involved in the transition from oxidized to reduced enzyme were not reported.…”

mentioning

confidence: 98%

“…It is a membrane-bound cytochrome bc complex composed of two subunits, NorC and NorB, that catalyzes the reduction of NO to N 2 O (5). The complex contains four known redox centers: three heme groups and one non-heme Fe atom (5)(6)(7). Heme c (six-coordinate, lowspin) is bound to NorC subunit and functions as the electron entry site of the enzyme.…”

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confidence: 99%

Nitric-oxide Reductase

Pinakoulaki¹,

Gemeinhardt²,

Saraste³

et al. 2002

Journal of Biological Chemistry

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We have applied resonance Raman spectroscopy to investigate the properties of the dinuclear center of oxidized, reduced, and NO-bound nitric-oxide reductase from Paracoccus denitrificans. , respectively. The nitrosyl species we detect is photolabile and can be photolyzed to generate a new form of oxidized enzyme in which the proximal histidine is ligated to heme b 3 , in contrast to the resting form. Photodissociation of the NO ligand yields a five-coordinate high-spin heme b 3 . Based on the findings reported here, the structure and properties of the dinuclear center of nitricoxide reductase in the oxidized, reduced, and NO-bound form as well as its photoproduct can be described with certainty.The bacterial nitric-oxide reductase (Nor) 1 complex forms the N-N bond during denitrification (1-4). It is a membrane-bound cytochrome bc complex composed of two subunits, NorC and NorB, that catalyzes the reduction of NO to N 2 O (5). The complex contains four known redox centers: three heme groups and one non-heme Fe atom (5-7). Heme c (six-coordinate, lowspin) is bound to NorC subunit and functions as the electron entry site of the enzyme. NorB contains one six-coordinate, low-spin heme b and a five-coordinate, high-spin heme b 3 , which, together with a non-heme iron atom, form the dinuclear NO reduction site. The sequence of NorB contains all six histidines that are the ligands of heme a, heme a 3 , and Cu B in cytochrome c oxidase (4).Resonance Raman (RR) scattering is a powerful technique for the study of heme proteins because the spectra are rich in information about the heme groups (8, 9). Moreover, vibrational modes of ligands bound to heme may be assigned by isotopic substitution measurements, and their properties, which reflect ligand structure, may be studied. It has been reported that in oxidized Nor, heme b 3 is not coordinated to the protein by its proximal histidine residue (6). In addition, the single oxygen isotope-sensitive ligand vibration observed at 811 cm Ϫ1 in the RR spectrum was attributed to the as (Fe-OFe) of the heme b 3 /non-heme diiron center (7). However, the structural implications involved in the transition from oxidized to reduced enzyme were not reported.The properties of oxidized Nor, when compared with those of the chemically generated deoxy (five-coordinate) and the nitrosyl complex, can be related to structural changes that take place in the protein upon ligand binding and release. It is these interactions between the heme and the protein that determine the biological properties of the heme protein. Moreover, in unraveling the NO protonation mechanism, it is necessary to establish sites in the enzyme that have exchangeable protons. If there are such sites near the high-spin heme b 3 or the non-heme Fe, then solvent exchange from protonated to deuterated buffers could lead to changes in the RR spectra because the vibrational frequencies are sensitive to effective mass.In an effort to gain information concerning these observations, we have used 413.1 nm RR excitation to examine the s...

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Nitric Oxide Reductase from Paracoccus denitrificans Contains an Oxo-Bridged Heme/Non-Heme Diiron Center

Cited by 90 publications

References 25 publications

Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin

Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin

Spectroscopic characterization of heme iron–nitrosyl species and their role in NO reductase mechanisms in diiron proteins

Nitric-oxide Reductase

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