Production and characterisation of Met80X mutants of yeast iso-1-cytochrome c: spectral, photochemical and binding studies on the ferrous derivatives

Silkstone, Gary; Stanway, Glyn; Brzezinski, Peter; Wilson, Michael T.

doi:10.1016/s0301-4622(02)00085-6

Cited by 34 publications

(59 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Mutagenesis-Site-directed mutagenesis, protein expression in Escherichia coli, and purification of yeast iso-1-cytochrome c mutants bearing single (M80A, M80S, M80D, or M80R) mutations in the heme environment were performed as described previously (8). The double mutations (M80D/F82D or M80D/ F82G) were introduced using a single-step PCR approach (8), and in addition using the M80D mutated pBPCYC1(wt)/3 plasmid construct as template DNA.…”

Section: Methodsmentioning

confidence: 99%

“…The double mutations (M80D/F82D or M80D/ F82G) were introduced using a single-step PCR approach (8), and in addition using the M80D mutated pBPCYC1(wt)/3 plasmid construct as template DNA. In these proteins, the Cys-102 residue is also changed to a Thr to prevent dimerization through formation of disulfide bridges.…”

Section: Methodsmentioning

confidence: 99%

“…Determination of Quantum Yield of CO Escape-The apparent quantum yield of dissociated CO on the millisecond time scale was also measured by the "pulsed" method developed by Brunori and co-workers (28) and as described previously (8). The method determines the value of for a given protein relative to a standard, generally Mb, for which the value is assigned as ϭ 1 (i.e.…”

Section: Methodsmentioning

confidence: 99%

“…native" states, we have made a series of Met-80 mutants of yeast iso-1-cytochrome c, namely M80A, M80S, M80T, M80D, and M80E (8). Such mutants bind CO (8,9).…”

mentioning

confidence: 99%

“…The replacement residues for the native Met-80 were selected as those that were not expected to coordinate to the heme iron, especially in the ferrous redox state. However, for electron transfer experiments, these mutants have one drawback, namely that the apparent quantum yield ( ) for photon-induced CO escape from the protein is very small (8). This is because of rapid and efficient "geminate" recombination of CO and the heme.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Ligand Dynamics in an Electron Transfer Protein

Silkstone

Jasaitis

Wilson

et al. 2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Substitution of the heme coordination residue Met-80 of the electron transport protein yeast iso-1-cytochrome c allows external ligands like CO to bind and thus increase the effective redox potential. This mutation, in principle, turns the protein into a quasi-native photoactivable electron donor. We have studied the kinetic and spectral characteristics of geminate recombination of heme and CO in a series of single M80X (X ‫؍‬ Ala, Ser, Asp, Arg) mutants, using femtosecond transient absorption spectroscopy. In these proteins, all geminate recombination occurs on the picosecond and early nanosecond time scale, in a multiphasic manner, in which heme relaxation takes place on the same time scale. The extent of geminate recombination varies from >99% (Ala, Ser) to ϳ70% (Arg), the latter value being in principle low enough for electron injection studies. The rates and extent of the CO geminate recombination phases are much higher than in functional ligand-binding proteins like myoglobin, presumably reflecting the rigid and hydrophobic properties of the heme environment, which are optimized for electron transfer. Thus, the dynamics of CO recombination in cytochrome c are a tool for studying the heme pocket, in a similar way as NO in myoglobin. We discuss the differences in the CO kinetics between the mutants in terms of the properties of the heme environment and strategies to enhance the CO escape yield. Experiments on double mutants in which Phe-82 is replaced by Asp or Gly as well as the M80D substitution indicate that such steric changes substantially increase the motional freedom-dissociated CO.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…native" states, we have made a series of Met-80 mutants of yeast iso-1-cytochrome c, namely M80A, M80S, M80T, M80D, and M80E (8). Such mutants bind CO (8,9).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Ligand Dynamics in an Electron Transfer Protein

Silkstone

Jasaitis

Wilson

et al. 2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

Converting Cytochrome c into a Peroxidase‐Like Metalloenzyme by Molecular Design

et al. 2007

View full text Add to dashboard Cite

Even better than the real thing. Based on molecular design, an electron‐transfer hemoprotein, cytochrome c, was converted into a peroxidase‐like enzyme by introduction of a distal histidine into the heme pocket. The cytochrome c variants obtained, Tyr67His and Tyr67His/Met80Val (see figure), showed much higher peroxidase activities than wild‐type cytochrome protein. More interestingly, the kcat/Km values of the new hemoproteins were higher than that of wild‐type horseradish peroxidase.

show abstract

Current Awareness on Yeast

2002

Yeast

View full text Add to dashboard Cite

In order to keep subscribers up‐to‐date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly‐published material on yeasts. Each bibliography is divided into 10 sections. 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (5 weeks journals ‐ search completed 2nd. Oct. 2002)

show abstract

Production and characterisation of Met80X mutants of yeast iso-1-cytochrome c: spectral, photochemical and binding studies on the ferrous derivatives

Cited by 34 publications

References 25 publications

Ligand Dynamics in an Electron Transfer Protein

Ligand Dynamics in an Electron Transfer Protein

Converting Cytochrome c into a Peroxidase‐Like Metalloenzyme by Molecular Design

Current Awareness on Yeast

Contact Info

Product

Resources

About