Exploring the terminal region of the proton pathway in the bacterial nitric oxide reductase

Flock, Ulrika; Lachmann, Peter; Reimann, Joachim; Watmough, Nicholas J.; Ädelroth, Pia

doi:10.1016/j.jinorgbio.2009.02.008

Cited by 34 publications

(42 citation statements)

References 26 publications

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“…Although two conserved glutamates (E198 and E267) are known to be crucial for NOR activity (24,25), their roles are not well defined (18,19). In a previous study (14), we demonstrated that one Glu, E68, is important for both iron binding and NOR activity of Fe B Mb.…”

Section: Discussion Using Rationally Designed Proteins To Address Impmentioning

confidence: 80%

“…4), suggesting that the second Glu may potentionally play a role in providing one of the protons during reduction of NO to N 2 O. Although free Glu outside a protein has a pK a ∼ 4.3, the studies of native NOR showed that the pK a of its Glu close to the active site has a value of ∼6.6 (22,25,33). The hydrogen binding network in our protein models may contribute to the fine-tuning of the Glu pK a to be more neutral, similar to that in NOR.…”

Section: Discussion Using Rationally Designed Proteins To Address Impmentioning

confidence: 99%

“…However, the metal center in HCOs is occupied by a copper (called Cu B ), whereas a nonheme iron is present in NOR (called Fe B ) (23,24). In addition, two conserved glutamates, shown by modeling to be close to the Fe B site (21, 22), are found to be essential for NOR activity (24,25). Some members of HCOs such as cytochrome cbb 3 oxidase display NOR activity (26-28), although the activity is ∼50-fold lower than native NOR (26).…”

mentioning

confidence: 99%

“…Some members of HCOs such as cytochrome cbb 3 oxidase display NOR activity (26-28), although the activity is ∼50-fold lower than native NOR (26). Therefore, it is important to elucidate the structural features, specifically the roles of the conserved glutamates close to the Fe B site and metal ions (copper vs. iron), responsible for the reduction of NO to N 2 O.To address these issues, biochemical and biophysical studies of native NOR and its variants have been carried out (24,25,(29)(30)(31)(32)(33)(34)(35)(36)(37). For example, Richardson and coworkers investigated the effects of amino acid substitutions of the five conserved glutamates (E122 and E125 presumed to face the periplasm and E198, E202, and E267 located in the interior of the membrane, close to the catalytic site) in the catalytic subunit of Paracoccus denitrificans, NorB.…”

mentioning

confidence: 99%

“…For example, Richardson and coworkers investigated the effects of amino acid substitutions of the five conserved glutamates (E122 and E125 presumed to face the periplasm and E198, E202, and E267 located in the interior of the membrane, close to the catalytic site) in the catalytic subunit of Paracoccus denitrificans, NorB. The E122A, E125A, E198A, and E267A variants were inactive, indicating that these four glutamates are crucial for NOR activity (24,25,32,33). On the other hand, Reimann et al constructed a 3D model of NorB using homology modeling with the structures of HCOs as templates and suggested a plausible pathway consisting of these conserved glutamates for proton delivery (22).…”

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confidence: 99%

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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.

108

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: Discussion Using Rationally Designed Proteins To Address Impmentioning

confidence: 80%

Section: Discussion Using Rationally Designed Proteins To Address Impmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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.

108

140

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Insights into the mechanism of nitric oxide reductase from a Fe_B‐depleted variant

et al. 2019

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A key step of denitrification, the reduction of toxic nitric oxide to nitrous oxide, is catalysed by cytochrome c‐dependent NO reductase (cNOR). cNOR contains four redox‐active cofactors: three hemes and a nonheme iron (FeB). Heme b3 and FeB constitute the active site, but the specific mechanism of NO‐binding events and reduction is under debate. Here, we used a recently constructed, fully folded and hemylated cNOR variant that lacks FeB to investigate the role of FeB during catalysis. We show that in the FeB‐less cNOR, binding of both NO and O2 to heme b3 still occurs but further reduction is impaired, although to a lesser degree for O2 than for NO. Implications for the catalytic mechanisms of cNOR are discussed.

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Rational Design of Protein Cages for Alternative Enzymatic Functions

Marshall

Miner

Wilson

et al. 2013

Coordination Chemistry in Protein Cages

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Exploring the terminal region of the proton pathway in the bacterial nitric oxide reductase

Cited by 34 publications

References 26 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

Insights into the mechanism of nitric oxide reductase from a Fe_B‐depleted variant

Rational Design of Protein Cages for Alternative Enzymatic Functions

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Exploring the terminal region of the proton pathway in the bacterial nitric oxide reductase

Cited by 34 publications

References 26 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

Insights into the mechanism of nitric oxide reductase from a FeB‐depleted variant

Rational Design of Protein Cages for Alternative Enzymatic Functions

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Product

Resources

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Insights into the mechanism of nitric oxide reductase from a Fe_B‐depleted variant