Determination of the Binding Interaction between Mitochondrial Electron Transport Chain Proteins Cytochrome C and Cytochrome C Oxidase

Abstract

The chromophore within green fluorescent protein (GFP) has a fluorescence quantum yield (FQY) three orders of magnitude greater than when free in solution. The dominant non-radiative decay process that competes with fluorescence is cis-trans isomerization, which simulations show to involve a redistribution of electron density (or charge transfer) between the chromophore's two conjugated rings. Two main hypotheses have been proposed to explain the increase in the chromophore's FQY within the protein environment. The first posits that the protein sterically confines the chromophore and prevents the bond rotation necessary for isomerization. The second suggests that the local electric field induced by the protein on the chromophore is oriented to oppose the charge transfer required for isomerization. Limited experimental evidence exists to interrogate the latter hypothesis. In this study, we introduce electron-donating and -withdrawing substituents on the chromophore's phenolate ring to systematically investigate the effects of sterics and electrostatics on energetic features of the chromophore's potential energy surface. Using amber suppression with substituted tyrosine residues in the model protein Dronpa2, we can incorporate substituents on the chromophore's phenolate ring by taking advantage of the chromophore's autocatalytic maturation process. Spectroscopic characterization of this library of protein variants allows for a direct comparison between a chromophore's steric and electronic properties and its energetic features in the ground and excited states, providing insight into the origins of GFP fluorescence. The methodology and conclusions from this work can be generalized to all chromophore-containing systems to provide a broad understanding of the factors that control fluorescence and isomerization with implications for protein design.