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A new approach to electron transfer: Semisynthetic cytochrome c
REDOX BIOCHEMISTRY
211
A NEW APPROACH TO ELECIRON TRANSFER: SEMISYNTHETIC CYTOCHROME C. A. L. Raohael and H. B. Gray, California Institute of Technology, Pasadena, CA 91125, USA. Semisynthetic techniques are being applied to construct new systems for electron-transfer studies. Replacements of the axial iron ligand of horse heart cytochrome c (Met-80 -> His, Asp, Leu) have been carried out. These derivatives indicate that axial ligand character and hydrophilicity are important in determining redox and spectral properties as well as protein folding. Semisynthesis allows us to modify the heme area or incorporate unnatural amino acids in order to examine in a systematic manner the roles of nuclear and electronic factors on the rates of long range electron transfer in proteins.
Go21
6022
THE EFFECTS OF CHARGED HEME POCKET MUTATIONS ON THE ELECTRON TRANSFER RATE AND REDUCTION POTENTlAL OF MYOGLOBIN. T F 7-a and H. B. Gray, Caltech 127-72, Pasadena, CA 91125, USA. The redox and electron transfer properties of site-directed mutants of human myoglobin in which valine68 (in Van der Waals contact with the heme) has been changed to aspartate (V68D) and glutamate (V68E) were studied. Although the heme midpoint potential for both proteins are equal(-135mV vs. NHE), the glutamate carboxyl in V68E is coordinated to the Fe(lll) heme whereas the aspartate carboxyl in V68D is not. In neither protein does a carboxyl oxygen coordinate to the Fe(ll) heme. From flash photolysis studies of the Ru(NH3)6-histidine48 derivatives of these proteins, the effect of Fe-O breaking upon the reorganization energy and rate of electron transfer was determined.
023
FERROCYTOCHROME B, REDUCTION OF FIZRRICYTOCHROME C. L. Eltis. P.D. Barker. & A.G. Mauk, Department of Biochemistry, University of British Columbia, Vancouver, British Columbia V6T 1W5 Canada. The kinetics of ferrocytochrome b, reduction of ferricytochrome c was sNdkd under a variety of solution conditions by anaerobic stopped-flow spectroscopy. At p = 0.5 M, pH 7.0, 25°C. the rate constant for this reaction was found to be 18.5 (f0.9) x 10’ M-k* and 30.6 (k1.8) x lo6 M“.s’ for horse heart and yeast iso-l-cytocbromes c, respectively. Under similar condidons, tbe rate constant for dimefhylester heme IX-s&stiNted ferrocytochrome b, reduction of horse heart ferricytochromc c was 16.5 (k0.6) x lo6 M%‘. Rate constants for each of these reactions showed a strong inverse dependence on ionic strength. The exact nature of this dependence differs for each reaction. The characterization of the structural requirements of bimolecular protein-protein electron transfer through analysis of specific mutants of both proteins on the kinetics of ferrocytochrome b, reduction of ferricytochrome c is discussed. (Supported by NIH Grant GM-28834).
211
A NEW APPROACH TO ELECIRON TRANSFER: SEMISYNTHETIC CYTOCHROME C. A. L. Raohael and H. B. Gray, California Institute of Technology, Pasadena, CA 91125, USA. Semisynthetic techniques are being applied to construct new systems for electron-transfer studies. Replacements of the axial iron ligand of horse heart cytochrome c (Met-80 -> His, Asp, Leu) have been carried out. These derivatives indicate that axial ligand character and hydrophilicity are important in determining redox and spectral properties as well as protein folding. Semisynthesis allows us to modify the heme area or incorporate unnatural amino acids in order to examine in a systematic manner the roles of nuclear and electronic factors on the rates of long range electron transfer in proteins.
Go21
6022
THE EFFECTS OF CHARGED HEME POCKET MUTATIONS ON THE ELECTRON TRANSFER RATE AND REDUCTION POTENTlAL OF MYOGLOBIN. T F 7-a and H. B. Gray, Caltech 127-72, Pasadena, CA 91125, USA. The redox and electron transfer properties of site-directed mutants of human myoglobin in which valine68 (in Van der Waals contact with the heme) has been changed to aspartate (V68D) and glutamate (V68E) were studied. Although the heme midpoint potential for both proteins are equal(-135mV vs. NHE), the glutamate carboxyl in V68E is coordinated to the Fe(lll) heme whereas the aspartate carboxyl in V68D is not. In neither protein does a carboxyl oxygen coordinate to the Fe(ll) heme. From flash photolysis studies of the Ru(NH3)6-histidine48 derivatives of these proteins, the effect of Fe-O breaking upon the reorganization energy and rate of electron transfer was determined.
023
FERROCYTOCHROME B, REDUCTION OF FIZRRICYTOCHROME C. L. Eltis. P.D. Barker. & A.G. Mauk, Department of Biochemistry, University of British Columbia, Vancouver, British Columbia V6T 1W5 Canada. The kinetics of ferrocytochrome b, reduction of ferricytochrome c was sNdkd under a variety of solution conditions by anaerobic stopped-flow spectroscopy. At p = 0.5 M, pH 7.0, 25°C. the rate constant for this reaction was found to be 18.5 (f0.9) x 10’ M-k* and 30.6 (k1.8) x lo6 M“.s’ for horse heart and yeast iso-l-cytocbromes c, respectively. Under similar condidons, tbe rate constant for dimefhylester heme IX-s&stiNted ferrocytochrome b, reduction of horse heart ferricytochromc c was 16.5 (k0.6) x lo6 M%‘. Rate constants for each of these reactions showed a strong inverse dependence on ionic strength. The exact nature of this dependence differs for each reaction. The characterization of the structural requirements of bimolecular protein-protein electron transfer through analysis of specific mutants of both proteins on the kinetics of ferrocytochrome b, reduction of ferricytochrome c is discussed. (Supported by NIH Grant GM-28834).