The pseudoazurin gene from Thiosphaera pantotropha: analysis of upstream putative regulatory sequences and overexpression in Escherichia coli

Leung, Yun Chung; Chan, Christopher; Reader, John S.; Willis, Anthony C.; Spanning, Rob J.M. van; Ferguson, Stuart J.; Radford, Sheena E.

doi:10.1042/bj3210699

Cited by 25 publications

(32 citation statements)

References 30 publications

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“…The protein sequence translated from the P. denitrificans pazS structural gene sequence was in exact agreement with that obtained by direct protein sequencing (25). These gene and protein sequences are very similar to their counterparts in the closely related organism (37) Paracoccus pantotrophus (11,25).…”

Section: Resultssupporting

confidence: 69%

“…These gene and protein sequences are very similar to their counterparts in the closely related organism (37) Paracoccus pantotrophus (11,25). However, immediately beyond the structural gene region the DNA sequences of P. pantotrophus and P. denitrificans had considerable differences.…”

Section: Resultsmentioning

confidence: 53%

“…2). Nevertheless, in both organisms the pazS gene clearly had an anaerobox of the Fnr type centered at bp 80.5 upstream of the translational initiation point and most likely at a similar position, bp 41.5, relative to the transcriptional start point, if the latter was assumed to be identically positioned in P. denitrificans and P. pantotrophus (6,25) (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

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A Mutant of Paracoccus denitrificans with Disrupted Genes Coding for Cytochrome c ₅₅₀ and Pseudoazurin Establishes These Two Proteins as the In Vivo Electron Donors to Cytochrome cd ₁ Nitrite Reductase

et al. 2003

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In Paracoccus denitrificans, electrons pass from the membrane-bound cytochrome bc 1 complex to the periplasmic nitrite reductase, cytochrome cd 1 . The periplasmic protein cytochrome c 550 has often been implicated in this electron transfer, but its absence, as a consequence of mutation, has previously been shown to result in almost no attenuation in the ability of the nitrite reductase to function in intact cells. Here, the hypothesis that cytochrome c 550 and pseudoazurin are alternative electron carriers from the cytochrome bc 1 complex to the nitrite reductase was tested by construction of mutants of P. denitrificans that are deficient in either pseudoazurin or both pseudoazurin and cytochrome c 550 . The latter organism, but not the former (which is almost indistinguishable in this respect from the wild type), grows poorly under anaerobic conditions with nitrate as an added electron acceptor and accumulates nitrite in the medium. Growth under aerobic conditions with either succinate or methanol as the carbon source is not significantly affected in mutants lacking either pseudoazurin or cytochrome c 550 or both these proteins. We concluded that pseudoazurin and cytochrome c 550 are the alternative electron mediator proteins between the cytochrome bc 1 complex and the cytochrome cd 1 -type nitrite reductase. We also concluded that expression of pseudoazurin is mainly controlled by the transcriptional activator FnrP.

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Section: Resultssupporting

confidence: 69%

Section: Resultsmentioning

confidence: 53%

Section: Resultsmentioning

confidence: 99%

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A Mutant of Paracoccus denitrificans with Disrupted Genes Coding for Cytochrome c ₅₅₀ and Pseudoazurin Establishes These Two Proteins as the In Vivo Electron Donors to Cytochrome cd ₁ Nitrite Reductase

et al. 2003

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“…Concentrations of oxidized Y25S cytochrome cd 1 were assayed spectroscopically using the absorbance at 410 nm (⑀ ϭ 255,000 M Ϫ1 cm Ϫ1 ). P. pantotrophus pseudoazurin was purified from Escherichia coli XL1-Blue (Stratagene) that contained the plasmid pJR2 (12). Concentrations of oxidized P. pantotrophus pseudoazurin were determined at 590 nm (⑀ ϭ 1360 M Ϫ1 cm Ϫ1 ).…”

Section: Methodsmentioning

confidence: 99%

Y25S Variant of Paracoccus pantotrophus Cytochrome cd1 Provides Insight into Anion Binding by d1 Heme and a Rare Example of a Critical Difference between Solution and Crystal Structures

Zajicek

Cheesman

Gordon

et al. 2005

Journal of Biological Chemistry

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Tyr25 is a ligand to the active site d 1 heme in as isolated, oxidized cytochrome cd 1 nitrite reductase from Paracoccus pantotrophus. This form of the enzyme requires reductive activation, a process that involves not only displacement of Tyr 25 from the d 1 heme but also switching of the ligands at the c heme from bis-histidinyl to His/Met. A Y25S variant retains this bis-histidinyl coordination in the crystal of the oxidized state that has sulfate bound to the d 1 heme iron. This Y25S form of the enzyme does not require reductive activation, an observation previously interpreted as meaning that the presence of the phenolate oxygen of Tyr 25 is the critical determinant of the requirement for activation. This interpretation now needs re-evaluation because, unexpectedly, the oxidized as prepared Y25S protein, unlike the wild type, has different heme iron ligands in solution at room temperature, as judged by magnetic circular dichroism and electron spin resonance spectroscopies, than in the crystal. In addition, the binding of nitrite and cyanide to oxidized Y25S cytochrome cd 1 is markedly different from the wild type enzyme, thus providing insight into the affinity of the oxidized d 1 heme ring for anions in the absence of the steric barrier presented by Tyr 25 .

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“…These cupredoxins are involved in denitrification processes in potent denitrifying bacteria, where their expression is enhanced when the host organism is grown under denitrifying conditions~Leung et al, 1997!. Pseudoazurins participate in a wide range of electron transport reactions and are able to interact with a number of structurally different proteins, including cytochrome cd1, Cu-NIR and cytochrome oxidase~Inoue et al, 1994; Williams et al, 1995;Kukimoto et al, 1996;Leung et al, 1997!.…”

Section: Pseudoazurin Subfamilymentioning

confidence: 99%

Blue copper proteins: A comparative analysis of their molecular interaction properties

et al. 2000

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Blue copper proteins are type-I copper-containing redox proteins whose role is to shuttle electrons from an electron donor to an electron acceptor in bacteria and plants. A large amount of experimental data is available on blue copper proteins; however, their functional characterization is hindered by the complexity of redox processes in biological systems. We describe here the application of a semiquantitative method based on a comparative analysis of molecular interaction fields to gain insights into the recognition properties of blue copper proteins. Molecular electrostatic and hydrophobic potentials were computed and compared for a set of 33 experimentally-determined structures of proteins from seven blue copper subfamilies, and the results were quantified by means of similarity indices. The analysis provides a classification of the blue copper proteins and shows that~1! comparison of the molecular electrostatic potentials provides useful information complementary to that highlighted by sequence analysis;~2! similarities in recognition properties can be detected for proteins belonging to different subfamilies, such as amicyanins and pseudoazurins, that may be isofunctional proteins;~3! dissimilarities in interaction properties, consistent with experimentally different binding specificities, may be observed between proteins belonging to the same subfamily, such as cyanobacterial and eukaryotic plastocyanins;~4! proteins with low sequence identity, such as azurins and pseudoazurins, can have sufficient similarity to bind to similar electron donors and acceptors while having different binding specificity profiles.Keywords: blue copper proteins; electrostatic potential; electron transfer; hydrophobic potential; protein-protein interactions; redox proteins; similarity index Blue copper proteins, which are also known as cupredoxins, are small, soluble proteins~10-14 kDa! whose active site contains a type-I copper. As far as it is known, they exert their function by shuttling electrons from a protein acting as an electron donor to another acting as an electron acceptor in various biological systems, such as bacterial and plant photosynthesis~Baker, 1994; Sykes, 1994!.A large amount of structural and spectroscopic data is available for the blue copper proteins, which have been named and classified into subfamilies according to their spectroscopic properties~Ad-man, 1991!. High resolution X-ray and NMR structures are known for several members of each of the plastocyanin, azurin, pseudoazurin, and amicyanin subfamilies~Baker, 1994!, and for single members of three further subfamilies, the rusticyanins~Harvey et al., 1998!, the cucumber basic proteins~CBP!~Guss et al., 1996!, and the stellacyanins~Hart et al., 1996!. Despite often showing low~Ͻ20%! sequence identity, all these proteins possess an eight-stranded Greek key b-barrel or b-sandwich fold and have a highly conserved active site architecture~Baker, 1994; Sykes, 1994!. Important information concerning the functional role and the binding properties of th...

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The pseudoazurin gene from Thiosphaera pantotropha: analysis of upstream putative regulatory sequences and overexpression in Escherichia coli

Cited by 25 publications

References 30 publications

A Mutant of Paracoccus denitrificans with Disrupted Genes Coding for Cytochrome c ₅₅₀ and Pseudoazurin Establishes These Two Proteins as the In Vivo Electron Donors to Cytochrome cd ₁ Nitrite Reductase

A Mutant of Paracoccus denitrificans with Disrupted Genes Coding for Cytochrome c ₅₅₀ and Pseudoazurin Establishes These Two Proteins as the In Vivo Electron Donors to Cytochrome cd ₁ Nitrite Reductase

Y25S Variant of Paracoccus pantotrophus Cytochrome cd1 Provides Insight into Anion Binding by d1 Heme and a Rare Example of a Critical Difference between Solution and Crystal Structures

Blue copper proteins: A comparative analysis of their molecular interaction properties

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The pseudoazurin gene from Thiosphaera pantotropha: analysis of upstream putative regulatory sequences and overexpression in Escherichia coli

Cited by 25 publications

References 30 publications

A Mutant of Paracoccus denitrificans with Disrupted Genes Coding for Cytochrome c 550 and Pseudoazurin Establishes These Two Proteins as the In Vivo Electron Donors to Cytochrome cd 1 Nitrite Reductase

A Mutant of Paracoccus denitrificans with Disrupted Genes Coding for Cytochrome c 550 and Pseudoazurin Establishes These Two Proteins as the In Vivo Electron Donors to Cytochrome cd 1 Nitrite Reductase

Y25S Variant of Paracoccus pantotrophus Cytochrome cd1 Provides Insight into Anion Binding by d1 Heme and a Rare Example of a Critical Difference between Solution and Crystal Structures

Blue copper proteins: A comparative analysis of their molecular interaction properties

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Product

Resources

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A Mutant of Paracoccus denitrificans with Disrupted Genes Coding for Cytochrome c ₅₅₀ and Pseudoazurin Establishes These Two Proteins as the In Vivo Electron Donors to Cytochrome cd ₁ Nitrite Reductase

A Mutant of Paracoccus denitrificans with Disrupted Genes Coding for Cytochrome c ₅₅₀ and Pseudoazurin Establishes These Two Proteins as the In Vivo Electron Donors to Cytochrome cd ₁ Nitrite Reductase