Semisynthesis of Axial-Ligand (Position 80) Mutants of Cytochrome c

Journal of the American Chemical Society

Volumn 113, Issue 3, 1991, Pages 1038-1040

  Raphael, Adrienne L ^a   Gray, Harry B ^a  

^a CALIFORNIA INSTITUTE OF TECHNOLOGY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

EID: 44749084371     PISSN: 00027863     EISSN: 15205126     Source Type: Journal    
DOI: 10.1021/ja00003a045     Document Type: Article

References (43)

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15. Syntheses involved reaction of 1–65 lactone (CNBr cleavage product of cytochrome c7 ) and peptide 66–104.7,13 Cyt c-Cys80 was synthesized by using 0.35 mM 1–65 lactone and peptide 66–104(Cys80) in 5 mM sodium phosphate buffer, pH 7.0, which was degassed and reduced with 4 equiv of sodium dithionite. After reaction under Ar for 36 h at ambient temperature with addition of 0.5 equiv of dithionite every 12 h, the products were purified by using cation-exchange chromatography (Pharmacia FPLC 10/10 Mono S column) with a 0.15–0.40 M NaCl gradient in 0.05 M sodium phosphate buffer, pH 7.0. Isoelectric focusing showed that cyt c-Cys80 was one species, with an isoelectric point equal to that of cytochrome c. Amino acid analyses agreed well with calculated values (in most cases within 2%).
16. Peptides 66–104 were synthesized by using a stepwise solid-phase method performed on an ABI 43A synthesizer. Kent, S. B. H. Annu. Rev. Biochem. 1988, 57, 957–985.
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19. Computer representation of the replacement of Met80 with cysteine and local energy minimization were carried out on the tuna cytochrome c structure, modified to the horse sequence (Takano, T.; Dickerson, R. E. J. Mol. Biol. 1981, 153, 79–94) by using BIOGRAF/III (Biodesign, Inc.).
20. For cyt c-Leu80, 0.07 mM 1–65 lactone and 66–104(Leu80) were combined in a two-compartment cell with medium-porosity frit in degassed 5 mM sodium phosphate solution; the counter-electrode solution contained 0.04 M NaCl and 0.56 mM [Ru(NH3)6]Cl3 in the same buffer. A potential of -450 mV vs SCE was applied for 16 h at ambient temperatures with stirring, using a Princeton Applied Research (PAR) Model 175 universal programmer in conjunction with a PAR Model 173 potentiostat/galvanostat. Platinum baskets were used as the working and counter electrodes. Products were purified as above (ref 12).
21. The overall reaction yields for the Cys, Leu, and His mutants were 37, 12, and 60%. Greater than 20 mg of the Cys and His derivatives can be synthesized conveniently.
22. Aspartate substitution was attempted by using a variety of synthetic conditions (0–0.2 M NaCl, pH 5–8, 22-50 °C, 24–48 h) in a procedure similar to that for cyt c-Cys80, but yields were less than 5%.
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25. The direct ligation of cysteine to Fe(III) in cyt c-Cys80 has been confirmed by using MCD spectroscopy: Rux, J.; Dawson, J. H., manuscript in preparation. The low yield of cyt c-Leu80 precluded MCD measurements.
26. DPP measurements were conducted by using a PAR Model 174A polarographic analyzer and gold disk electrode on 0.2 mM protein solutions containing 0.01 M 4,4′-bipyridine, 0.2 M NaCl in 0.05 M sodium phosphate pH 7.0 (25 °C). Electrode potentials were measured with a Keithley 177 microvoltmeter, relative to SCE reference. Results were recorded on a Houston Instruments Omnigraphics 2000 recorder.
27. Spectroelectrochemical determination of reduction potentials was carried out at 22 °C in 0.2 M NaCl and 0.05 M sodium phosphate buffer, pH 6.8, under anaerobic conditions. For procedures, see: Ellis, W. R., Jr.; Wang, H.; Blair, D. F.; Gray, H. B.; Chan, S. I. Biochemistry 1986, 25, 161–167. [Ru(NH3)6]Cl3 (cyt c-Leu80) and anthraquinone-2-sulfonate (cyt c-Cys80) were used as mediators.
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