The cytochrome cbb3 from Pseudomonas stutzeri displays nitric oxide reductase activity

Forte, Elena; Urbani, Andrea; Saraste, Matti; Sarti, Paolo; Brunori, Maurizio; Giuffrè, Alessandro

doi:10.1046/j.0014-2956.2001.02597.x

Cited by 112 publications

(117 citation statements)

References 28 publications

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“…5). Significantly, a fivecoordinate heme-NO species has also been observed for both NOR (30,31,35) and the member of the HCO family with the highest NO reduction activity, cytochrome cbb 3 oxidases (26,62). However, this species was not observed for FeðIIÞ-Fe B Mb-NO and CuðIÞ-Fe B Mb-NO, which lack the second Glu (E107).…”

Section: Discussion Using Rationally Designed Proteins To Address Impmentioning

confidence: 91%

“…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. Moreover, it is interesting that Cu(I)-I107E Fe B Mb also shows NOR activity, which provides an interesting protein model of HCOs with NOR function (26)(27)(28), even though Glu residues are not conserved in native HCOs. Additionally, spectroelectrochemical studies showed that the reduction potential of I107E Fe B Mb with no metal ion in the Fe B site is similar to that of Fe B Mb, but much lower than that of Cu B Mb (77 mV) (54), which contains the same three His, but no Glu in the metal-binding site above the heme.…”

Section: Discussion Using Rationally Designed Proteins To Address Impmentioning

confidence: 99%

“…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)(27)(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.…”

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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.

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: Discussion Using Rationally Designed Proteins To Address Impmentioning

confidence: 91%

Section: Discussion Using Rationally Designed Proteins To Address Impmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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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“…Among the O 2 -reducing HCuOs, the cbb 3 type is the closest relative to NOR, and interestingly, cbb 3 -type oxidases, in contrast to A-type, have substantial NO-reduction activities (9,10). The A-type oxidases contain, in their catalytic subunit I, a lowspin heme a and a high-spin heme a 3 -Cu B active site.…”

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confidence: 99%

Entrance of the proton pathway in cbb ₃ -type heme-copper oxidases

Lee

Gennis²,

Ädelroth

2011

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

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Heme-copper oxidases (HCuOs) are the last components of the respiratory chain in mitochondria and many bacteria. They catalyze O 2 reduction and couple it to the maintenance of a proton-motive force across the membrane in which they are embedded. In the mitochondrial-like, A family of HCuOs, there are two well established proton transfer pathways leading from the cytosol to the active site, the D and the K pathways. In the C family (cbb 3 ) HCuOs, recent work indicated the use of only one pathway, analogous to the K pathway. In this work, we have studied the functional importance of the suggested entry point of this pathway, the Glu-25 (Rhodobacter sphaeroides cbb 3 numbering) in the accessory subunit CcoP (E25 P ). We show that catalytic turnover is severely slowed in variants lacking the protonatable Glu-25. Furthermore, proton uptake from solution during oxidation of the fully reduced cbb 3 by O 2 is specifically and severely impaired when Glu-25 was exchanged for Ala or Gln, with rate constants 100-500 times slower than in wild type. Thus, our results support the role of E25 P as the entry point to the proton pathway in cbb 3 and that this pathway is the main proton pathway. This is in contrast to the A-type HCuOs, where the D (and not the K) pathway is used during O 2 reduction. The cbb 3 is in addition to O 2 reduction capable of NO reduction, an activity that was largely retained in the E25 P variants, consistent with a scenario where NO reduction in cbb 3 uses protons from the periplasmic side of the membrane.glutamate | nitric oxide | oxygen reduction

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“…14 However, they show a close relationship to the nitric oxide reductases (NORs), 15 another distant member of the HCO superfamily, 16 and, interestingly, also display NOR activity in vitro. 17 Like other members of the HCO superfamily, cbb 3 -CcOs comprise a central catalytic subunit (CcoN) consisting of 12 transmembrane helices. 16 Besides CcoN (52.8 kDa), the cbb 3 -CcO from P. stutzeri ZoBell, previously characterized in molecular, spectroscopic, and structural aspects by Pitcher et al, 18,19 contains the transmembrane subunits CcoO (22.9 kDa) and CcoP (34 kDa) that harbor one and two heme c molecules, respectively.…”

Section: Introductionmentioning

confidence: 99%

A decade of crystallization drops: Crystallization of the cbb₃ cytochrome c oxidase from Pseudomonas stutzeri

et al. 2014

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The cbb 3 cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O 2 reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb 3 oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O 2 reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb 3 oxidase isoenzymes. The exchange of n-dodecyl-b-D-maltoside for a precisely defined mixture of two a-maltosides and decanoylsucrose as well as the choice of the crystallization method had a most profound impact on crystal quality. This report highlights problems frequently encountered in membrane protein crystallization and offers meaningful approaches to improve crystal quality.

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The cytochrome cbb₃ from Pseudomonas stutzeri displays nitric oxide reductase activity

Cited by 112 publications

References 28 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

Entrance of the proton pathway in cbb ₃ -type heme-copper oxidases

A decade of crystallization drops: Crystallization of the cbb₃ cytochrome c oxidase from Pseudomonas stutzeri

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The cytochrome cbb3 from Pseudomonas stutzeri displays nitric oxide reductase activity

Cited by 112 publications

References 28 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

Entrance of the proton pathway in cbb 3 -type heme-copper oxidases

A decade of crystallization drops: Crystallization of the cbb3 cytochrome c oxidase from Pseudomonas stutzeri

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The cytochrome cbb₃ from Pseudomonas stutzeri displays nitric oxide reductase activity

Entrance of the proton pathway in cbb ₃ -type heme-copper oxidases

A decade of crystallization drops: Crystallization of the cbb₃ cytochrome c oxidase from Pseudomonas stutzeri