The Cytochrome c Domain of Dimeric Cytochrome cd1 of Paracoccus pantotrophus Can Be Produced at High Levels as a Monomeric Holoprotein Using an Improved c-Type Cytochrome Expression System in Escherichia coli

Gordon, Euan; Steensma, E.; Ferguson, Stuart J.

doi:10.1006/bbrc.2001.4425

Cited by 25 publications

(20 citation statements)

References 32 publications

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“…In contrast to holocytochrome cd 1 , this c-domain protein has His/Met (M106) ligation at the c heme in both the oxidized and reduced forms, as shown by NMR spectroscopy (35). A redox titration of this protein gave a midpoint potential of +250 mV (as expected for a His/Met coordinated heme) (18). In the present work, a M106H variant of the c-domain was subsequently made and expressed, using the method of Steensma et al (2001), and the reduction potential of the c heme was investigated.…”

Section: Resultsmentioning

confidence: 89%

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Probing the Unusual Oxidation/Reduction Behavior ofParacoccus pantotrophusCytochromecd₁Nitrite Reductase by Replacing a Switchable Methionine Heme Iron Ligand with Histidine

2006

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A M106H variant, where M106 is a c-type heme iron axial ligand, of cytochrome cd(1) nitrite reductase is an inactive protein in vivo. Expression of the holoprotein in Paracoccus pantotrophus required generation of nitric oxide during cell growth through simultaneous expression of an exogenous copper nitrite reductase from Alcaligenes faecalis. In the absence of the latter protein, only a semi-apo form of M106H cytochrome cd(1) was formed. Thus it was demonstrated that expression of the chromosomal nir genes for d(1) heme biosynthesis in P. pantotrophus is NO-dependent, probably mediated by the transcription factor NNR, and a route to low or zero activity mutants had been established. The value of such variants for mechanistic studies on cytochrome cd(1) is illustrated by the use of M106H to demonstrate that the d(1) heme potential can be resolved and measured at approximately +175 mV with the c heme shifted to -60 mV, consistent with its bishistidinyl coordination. The unusual highly cooperative and strongly hysteretic redox titration of the wild type is lost in the M106H protein. The same c heme midpoint potential was observed in a M106H variant of a c-domain construct. The difference between d(1) heme and c heme redox potentials has allowed preparation of a M106H protein with oxidized c heme and reduced d(1) heme. This one electron reduced form will reduce nitrite to nitric oxide, but the latter remains bound to the resulting fully oxidized enzyme.

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Section: Resultsmentioning

confidence: 89%

“…Previously, Steensma et al (35) have shown that it is possible to express a truncated cytochrome cd 1 which contains only the domain binding of the c heme (18). In contrast to holocytochrome cd 1 , this c-domain protein has His/Met (M106) ligation at the c heme in both the oxidized and reduced forms, as shown by NMR spectroscopy (35).…”

Section: Resultsmentioning

confidence: 98%

Probing the Unusual Oxidation/Reduction Behavior ofParacoccus pantotrophusCytochromecd₁Nitrite Reductase by Replacing a Switchable Methionine Heme Iron Ligand with Histidine

2006

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“…Briefly, the proteins were produced in Escherichia coli strain JCB7123 (Gordon et al, 2001), except for domain AB that was produced in BL21(DE3). Both expression strains were co-transformed with cytochrome maturation plasmid pEC86 (Arslan et al, 1998).…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Structure of a novel dodecaheme cytochrome c from Geobacter sulfurreducens reveals an extended 12nm protein with interacting hemes

Pokkuluri

Londer

Duke

et al. 2011

Journal of Structural Biology

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“…In these conditions, natural reductants of c-type cytochromes are thought to be absent and the expression system therefore probes the oxidation state of the end product of the cytochrome c biogenesis apparatus. Both the c heme binding domain of P. pantotrophus cytochrome cd 1 [27] and the semi-apo form of cd 1 (which contains no d 1 heme) (RSZ and SJF, unpublished data) are obtained in the reduced state when produced in this way. Similarly, our recent work has shown that in vitro the characteristic covalent attachment of heme to c-type cytochromes requires reducing conditions [28,29].…”

Section: Discussionmentioning

confidence: 99%

Paracoccus pantotrophus NapC can reductively activate cytochrome cd1 nitrite reductase

ZAJICEK¹

2004

FEBS Letters

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The oxidized ''as isolated'' form of Paracoccus pantotrophus cytochrome cd 1 nitrite reductase has a bis-histidinyl coordinated c heme and a histidine/tyrosine coordinated d 1 heme. This form of the enzyme has previously been shown to be kinetically incompetent. Upon reduction, the coordination of both hemes changes and the enzyme is kinetically activated. Here, we show that P. pantotrophus NapC, a tetraheme c-type cytochrome belonging to a large family of such proteins, is capable of reducing, and hence activating, ''as isolated'' cytochrome cd 1 . NapC is the first protein from P. pantotrophus identified as being capable of this activation step and, given the periplasmic co-location and co-expression of the two proteins, is a strong candidate to be a physiological activation partner.

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The Cytochrome c Domain of Dimeric Cytochrome cd1 of Paracoccus pantotrophus Can Be Produced at High Levels as a Monomeric Holoprotein Using an Improved c-Type Cytochrome Expression System in Escherichia coli

Cited by 25 publications

References 32 publications

Probing the Unusual Oxidation/Reduction Behavior ofParacoccus pantotrophusCytochromecd₁Nitrite Reductase by Replacing a Switchable Methionine Heme Iron Ligand with Histidine

Probing the Unusual Oxidation/Reduction Behavior ofParacoccus pantotrophusCytochromecd₁Nitrite Reductase by Replacing a Switchable Methionine Heme Iron Ligand with Histidine

Structure of a novel dodecaheme cytochrome c from Geobacter sulfurreducens reveals an extended 12nm protein with interacting hemes

Paracoccus pantotrophus NapC can reductively activate cytochrome cd1 nitrite reductase

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The Cytochrome c Domain of Dimeric Cytochrome cd1 of Paracoccus pantotrophus Can Be Produced at High Levels as a Monomeric Holoprotein Using an Improved c-Type Cytochrome Expression System in Escherichia coli

Cited by 25 publications

References 32 publications

Probing the Unusual Oxidation/Reduction Behavior ofParacoccus pantotrophusCytochromecd1Nitrite Reductase by Replacing a Switchable Methionine Heme Iron Ligand with Histidine

Probing the Unusual Oxidation/Reduction Behavior ofParacoccus pantotrophusCytochromecd1Nitrite Reductase by Replacing a Switchable Methionine Heme Iron Ligand with Histidine

Structure of a novel dodecaheme cytochrome c from Geobacter sulfurreducens reveals an extended 12nm protein with interacting hemes

Paracoccus pantotrophus NapC can reductively activate cytochrome cd1 nitrite reductase

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Resources

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Probing the Unusual Oxidation/Reduction Behavior ofParacoccus pantotrophusCytochromecd₁Nitrite Reductase by Replacing a Switchable Methionine Heme Iron Ligand with Histidine

Probing the Unusual Oxidation/Reduction Behavior ofParacoccus pantotrophusCytochromecd₁Nitrite Reductase by Replacing a Switchable Methionine Heme Iron Ligand with Histidine