Improved system for heterologous expression of cytochrome c mutants in<i>Escherichia coli</i>

Tenger, Katalin; Khoroshyy, Petro; Kovács, Kornél L.; Zimányi, László; Rákhely, Gábor

doi:10.1556/abiol.58.2007.suppl.3

Cited by 7 publications

(3 citation statements)

References 12 publications

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“…All the horse mitochondrial cytochromes c produced in the current work by HCCS functioning in E. coli had normal UV–vis spectra (except of course the G6A and F10A variants). Others have shown that K8C, K8E and V11C variants of horse cytochrome c are matured by yeast HCCS [24,25].…”

Section: Resultsmentioning

confidence: 99%

The mitochondrial cytochromecN-terminal region is critical for maturation by holocytochromecsynthase

et al. 2011

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a b s t r a c tThe covalent attachment of heme to mitochondrial cytochrome c is catalysed by holocytochrome c synthase (HCCS, also called heme lyase). How HCCS functions and recognises the substrate apocytochrome is unknown. Here we have examined HCCS recognition of a chimeric substrate comprising a short mitochondrial cytochrome c N-terminal region with the C-terminal sequence, including the CXXCH heme-binding motif, of a bacterial cytochrome c that is not otherwise processed by HCCS. Heme attachment to the chimera demonstrates the importance of the N-terminal region of the cytochrome. A series of variants of a mitochondrial cytochrome c with amino acid replacements in the N-terminal region have narrowed down the specificity determinants, providing insight into HCCS substrate recognition.

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Section: Resultsmentioning

confidence: 99%

The mitochondrial cytochromecN-terminal region is critical for maturation by holocytochromecsynthase

et al. 2011

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“…The K87C mutant of horse heart cytochrome c was produced by site-directed mutagenesis and coexpression with the yeast cytochrome c heme lyase in the BL21-AI (Invitrogen) E. coli strain, according to procedures previously described. , The genes of the mutant cytochrome c and heme lyase were cloned into the arabinose-inducible pBAD24 plasmid. The successful mutation of the cytochrome gene has been verified by sequencing.…”

Section: Experimental Sectionmentioning

confidence: 99%

Disentangling Electron Tunneling and Protein Dynamics of Cytochrome c through a Rationally Designed Surface Mutation

et al. 2013

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Nonexponential distance dependence of the apparent electron-transfer (ET) rate has been reported for a variety of redox proteins immobilized on biocompatible electrodes, thus posing a physicochemical challenge of possible physiological relevance. We have recently proposed that this behavior may arise not only from the structural and dynamical complexity of the redox proteins but also from their interplay with strong electric fields present in the experimental setups and in vivo (J. Am Chem. Soc. 2010, 132, 5769-5778). Therefore, protein dynamics are finely controlled by the energetics of both specific contacts and the interaction between the protein's dipole moment and the interfacial electric fields. In turn, protein dynamics may govern electron-transfer kinetics through reorientation from low to high donor-acceptor electronic coupling orientations. Here we present a combined computational and experimental study of WT cytochrome c and the surface mutant K87C adsorbed on electrodes coated with self-assembled monolayers (SAMs) of varying thickness (i.e., variable strength of the interfacial electric field). Replacement of the positively charged K87 by a neutral amino acid allowed us to disentangle protein dynamics and electron tunneling from the reaction kinetics and to rationalize the anomalous distance dependence in terms of (at least) two populations of distinct average electronic couplings. Thus, it was possible to recover the exponential distance dependence expected from ET theory. These results pave the way for gaining further insight into the parameters that control protein electron transfer.

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“…Mutation of Gly6 and Phe10 to Ala in the horse cytochrome c or the yeast Cyc1p eliminates CCHL-dependent holocytochrome c production [27–28], underlining their critical role for CCHL activity. In the contrary, mutation of Gln16 in Cyc7p [32] or Gln15 in horse cytochrome c [24] to Cys does not hinder the ability of CCHL to mature the corresponding cytochrome c variants. Similar flexibility for equivalent amino acid residues within the heme binding site has also been observed for Ccm system I and its apocytochromes c substrates [42].…”

Section: Resultsmentioning

confidence: 99%

Engineering a prokaryotic apocytochrome c as an efficient substrate for Saccharomyces cerevisiae cytochrome c heme lyase

Verissimo

Sanders

Daldal

et al. 2012

Biochemical and Biophysical Research Communications

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Cytochromes c are heme proteins that require multiple maturation components, such as heme lyases, for cofactor incorporation. Saccharomyces cerevisiae has two heme lyases that are specific for apocytochromes c (CCHL) or c1 (CC1HL). CCHL can covalently attach heme b groups to apocytochrome c substrates of eukaryotic but not prokaryotic origin. Besides their conserved Cys-Xxx-Xxx-Cys-His heme-binding motifs, the amino-terminal regions of apocytochrome c substrates appear to be important for CCHL function. In this study, we show for the first time that only two amino acid changes in the amino-terminal region of the non-CCHL substrate apocytochrome c2 from Rhodobacter capsulatus are necessary and sufficient for efficient holocytochrome c formation by CCHL. This finding led us to propose a consensus sequence located at the amino-terminus of apocytochromes c and critical for substrate recognition and heme ligation by CCHL.

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Improved system for heterologous expression of cytochrome c mutants inEscherichia coli

Cited by 7 publications

References 12 publications

The mitochondrial cytochromecN-terminal region is critical for maturation by holocytochromecsynthase

The mitochondrial cytochromecN-terminal region is critical for maturation by holocytochromecsynthase

Disentangling Electron Tunneling and Protein Dynamics of Cytochrome c through a Rationally Designed Surface Mutation

Engineering a prokaryotic apocytochrome c as an efficient substrate for Saccharomyces cerevisiae cytochrome c heme lyase

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