The Acidic Nature of the CcmG Redox-Active Center Is Important for Cytochrome<i>c</i>Maturation in<i>Escherichia coli</i>

Edeling, Melissa A.; Ahuja, Umesh; Heras, Begoña; Thöny‐Meyer, Linda; Martin, Jennifer L.

doi:10.1128/jb.186.12.4030-4033.2004

Cited by 21 publications

(21 citation statements)

References 31 publications

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“…Hence, it is unlikely that E80 has the same function as Asp 26 in thioredoxin. Consistent with this is the observation that substitution of Glu 86 in CcmG did not affect cytochrome c maturation (45).…”

Section: Glu 80 Plays a Key Role In Controlling The Reactivities Of Tsupporting

confidence: 63%

“…This is believed to deprotonate the second cysteine thiol of the active site, thus facilitating resolution of mixed disulfide intermediates (43,44). This raises the possibility that Glu 80 serves a similar function in ResA/CcmG homologues (45). D26A thioredoxin, however, was found to have significantly lower activity and an increased pK a value associated with its N-terminal active site thiol (46).…”

Section: Glu 80 Plays a Key Role In Controlling The Reactivities Of Tmentioning

confidence: 99%

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Molecular Basis for Specificity of the Extracytoplasmic Thioredoxin ResA

Lewin

Crow

Oubrie

et al. 2006

Journal of Biological Chemistry

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ResA, an extracytoplasmic thioredoxin from Bacillus subtilis, acts in cytochrome c maturation by reducing the disulfide bond present in apocytochromes prior to covalent attachment of heme. This reaction is (and has to be) specific, as broad substrate specificity would result in unproductive shortcircuiting with the general oxidizing thioredoxin(s) present in the same compartment. Using mutational analysis and subsequent biochemical and structural characterization of active site variants, we show that reduced ResA displays unusually low reactivity at neutral pH, consistent with the observed high pK a values >8 for both active site cysteines. Residue Glu 80 is shown to play a key role in controlling the acid-base properties of the active site. A model in which substrate binding dramatically enhances the reactivity of the active site cysteines is proposed to account for the specificity of the protein. Such a substratemediated activation mechanism is likely to have wide relevance for extracytoplasmic thioredoxins.

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Section: Glu 80 Plays a Key Role In Controlling The Reactivities Of Tsupporting

confidence: 63%

Section: Glu 80 Plays a Key Role In Controlling The Reactivities Of Tmentioning

confidence: 99%

Molecular Basis for Specificity of the Extracytoplasmic Thioredoxin ResA

Lewin

Crow

Oubrie

et al. 2006

Journal of Biological Chemistry

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“…However, previous studies of E. coli CcmG in which the equivalent glutamate residue (Glu86) was replaced by alanine revealed levels of mature cytochrome c that were comparable to those in the wild-type strain (14). However, CcmG is not the terminal reductase in system I CCM; i.e., it occurs earlier in the transfer chain, accepting electrons from the transmembrane DsbD protein and passing them on to CcmH (39).…”

Section: Replacement Of the N-terminal Transmembrane Anchor Ofmentioning

confidence: 92%

The Active-Site Cysteinyls and Hydrophobic Cavity Residues of ResA Are Important for CytochromecMaturation inBacillus subtilis

et al. 2008

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ResA is an extracytoplasmic membrane-bound thiol-disulfide oxidoreductase required for cytochrome c maturation in Bacillus subtilis. Previous biochemical and structural studies have revealed that the active-site cysteinyls cycle between oxidized and reduced states with a low reduction potential and that, upon reduction, a hydrophobic cavity forms close to the active site. Here we report in vivo studies of ResA-deficient B. subtilis complemented with a series of ResA variants. Using a range of methods to analyze the cellular cytochrome c content, we demonstrated (i) that the N-terminal transmembrane segment of ResA serves principally to anchor the protein to the cytoplasmic membrane but also plays a role in mediating the activity of the protein; (ii) that the active-site cysteines are important for cytochrome c maturation activity; (iii) that Pro141, which forms part of the hydrophobic cavity and which adopts a cis conformation, plays an important role in protein stability; (iv) that Glu80, which lies at the base of the hydrophobic cavity, is important for cytochrome c maturation activity; and, finally, (v) that Pro141 and Glu80 ResA mutant variants promote selective maturation of low levels of one c-type cytochrome, subunit II of the cytochrome c oxidase caa 3 , indicating that this apocytochrome is distinct from the other three endogenous c-type cytochromes of B. subtilis.

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“…On the other hand, it is plausible that Pa-CcmG recognition may be mediated by some conserved basic/polar residues located near the active site of Pa-CcmH* (i.e. Arg 24 and Gln 29 ); indeed, the unusually acidic nature of the redox-active center of E. coli CcmG has been hypothesized to be important in the interaction with its CcmH partner (17,18).…”

Section: Discussionmentioning

confidence: 99%

“…According to this view, the mixed disulfide complex between CcmH and apocyt is subsequently resolved by CcmG, releasing reduced apocyt and oxidized CcmH (15,16). Although CcmG is a structurally well characterized TRX-like protein that receives electrons from DsbD (17)(18)(19), no structural information is available for any CcmH protein. Previous studies indicated that CcmH is a membrane-bound protein exposing the conserved redox-active LRCXXCQ motif to the periplasm; its essential role in the maturation of apocyt was suggested by the observation that loss of CcmH impairs the synthesis of c-type cytochromes in vivo (13,14,20).…”

mentioning

confidence: 99%