Rates and Energetics of Tyrosine Ring Flips in Yeast Iso-2-cytochrome c

Barry T. Nall, Efrain H. Zuniga

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19 Scopus citations


Isotope-edited nuclear magnetic resonance spectroscopy is used to monitor ring flip motion of the five tyrosine side chains in the oxidized and reduced forms of yeast iso-2-cytochrome c. With specifically labeled protein purified from yeast grown on media containing [3,5-13C]tyrosine, isotope-edited one-dimensional proton spectra have been collected over a 5-55 °C temperature range. The spectra allow selective observation of the 10 3,5 tyrosine ring proton resonances and, using a two-site exchange model, allow estimation of the temperature dependence of ring flip rates from motion-induced changes in proton line shapes. For the reduced protein, tyrosines II and IV are in fast exchange throughout the temperature range investigated, or lack resolvable differences in static chemical shifts for the 3,5 ring protons. Tyrosines I, III, and V are in slow exchange at low temperatures and in fast exchange at high temperatures. Spectral simulations give flip rates for individual tyrosines in a range of one flip per second at low temperatures to thousands of flips per second at high temperatures. Eyring plots show that two of the tyrosines (I and III) have essentially the same activation parameters: ΔH* = 28 kcal/mol for both I and III; ΔS* = 42 cal/(mol•K) for I, and ΔS* = 41 cal/(mol•K) for III. The remaining tyrosine (V) has a larger enthalpy and entropy of activation: ΔH* = 36 kcal/mol, ΔS* = 72 cal/(mol•K). Tentative sequence-specific assignments for the tyrosines in reduced iso-2 are suggested by comparison to horse cytochrome c. For oxidized iso-2, five resonances are observed at high temperatures, suggesting flip rates for all five tyrosines sufficient to average static chemical shift differences. At lower temperatures, there is evidence of intermediate and slow flipping for some of the rings, but spectral simulations have not been possible because of the complexity of the isotope-edited spectra.

Original languageEnglish (US)
Pages (from-to)7576-7584
Number of pages9
Issue number33
StatePublished - Aug 1 1990

ASJC Scopus subject areas

  • Biochemistry


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