Derivatization of yeast cytochrome c peroxidase with pentaammineruthenium(III).
Cytochrome c peroxidase (CCP) was derivatized using aquopentaammineruthenium(II) [a5RuIIH2O] resulting in stable, covalently-linked derivatives that were purified by cation-exchange FPLC. Spectrophotometric determination of a5RuHis:heme ratios allowed identification of two derivatives containing one a5RuHis per CCP molecule. The histidine-specific reagent, diethyl pyrocarbonate (DEPC), which reacted with three histidine residues in native CCP (6, 60, 96) at pH 7, reacted with only two histidines in both a5RuHisCCP species. X-ray crystallography showed that a5Ru is coordinated to His60 in one derivative [Fox et al. (1990) J. Am. Chem. Soc. 112, 7426]; HPLC and mass spectral analysis of the tryptic peptides of the other derivative identified a peptide (MW = 1469 Da) corresponding to residues 1-12 of CCP plus a5Ru, indicating His6 as the site of modification. Mass spectral analysis of native CCP, a5RuHis60CCP, and the a5RuHis6 derivative yielded MWs of 33,536, 33,717, and 33,901 Da, respectively, revealing that a second site is ruthenated in the His6 derivative. Mass spectral analysis of a shoulder separated from the a5RuHis60CCP FPLC peak also indicated the presence of CCP with bound a5Ru (MW = 33,718 Da). Differential pulse voltammetry of this shoulder, which has negligible a5RuHis absorption, gave a peak at -68 mV (vs NHE) which is in the range expected for reduction of a5RuIII (carboxylato) complexes, as well as a peak at 42 mV due to the presence of approximately 20% a5RuHis60CCP. The extent of ruthenation at sites other than histidine was unexpected and illustrates that a5RuIIH2O is less specific for histidine than previously thought. Activity measurements and stability of enzyme intermediates were measured to further characterize the a5RuCCP species and showed that the derivatives have similar properties to native CCP.
Fox
T
T
Department of Chemistry and Biochemistry, Concordia University, Montreal, Québec, Canada.
English
A M
AM
Gibbs
B F
BF
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000302:isolation & purification; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D004563:Electrochemistry; D004795:Enzyme Stability; D006639:Histidine / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D017975:Ruthenium Compounds / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet
D017975:Ruthenium Compounds; D006639:Histidine; C063246:pentaammineruthenium(3+); D003578:Cytochrome-c Peroxidase; D004047:Diethyl Pyrocarbonate