The reaction between cytochrome c (Cc) and cytochrome c oxidase (CcO) was studied using horse cytochrome c derivatives labeled with ruthenium trisbipyridine at Cys 39 (Ru-39-Cc). Flash photolysis of a 1:1 complex between Ru-39-Cc and bovine CcO at a low ionic strength resulted in the electron transfer from photoreduced heme c to Cu A with an intracomplex rate constant of k 3 = 6 × 10 4 s −1 . The K13A, K72A, K86A, and K87A Ru-39-Cc mutants had nearly the same k 3 value but bound much more weakly to bovine CcO than wild-type Ru-39-Cc, indicating that lysines 13, 72, 86, and 87 were involved in electrostatic binding to CcO, but were not involved in the electron transfer pathway. The Rhodobacter sphaeroides (Rs) W143F mutant (bovine W104) caused a 450-fold decrease in k 3 but did not affect the binding strength with CcO or the redox potential of Cu A . These results are consistent with a computational model for Cc−CcO (Roberts and Pique (1999) J. Biol. Chem. 274, 38051−38060) with the following electron transfer pathway: heme c → CcO-W104 → CcO-M207 → Cu A . A crystal structure for the Cc−CcO complex with the proposed electron transfer pathway heme c → Cc-C14 → Cc-K13 → CcO-Y105 → CcO-M207 → Cu A (S. Shimada et al. (2017) EMBO J. 36, 291−300) is not consistent with the kinetic results because the K13A mutation had no effect on k 3 . Addition of 40% ethylene glycol (as present during the crystal preparation) decreased k 3 significantly, indicating that it affected the conformation of the complex. This may explain the discrepancy between the current results and the crystallographic structure.

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.

modes, and find that organizational changes can optimize for different objectives. Cargos with clustered motors are transported efficiently, but are slow to bind to microtubules. Cargos with motors dispersed rigidly on their surface bind microtubules quickly, but are transported inefficiently. Cargos with freelydiffusing motors have both fast binding and efficient transport, although less efficient than clustered motors. These results point to a functional role for observed changes in motor organization on cargos, and suggest motor diffusivity as a control point for transport, either by modulation of adaptor proteins or changes in lipid composition.

modes, and find that organizational changes can optimize for different objectives. Cargos with clustered motors are transported efficiently, but are slow to bind to microtubules. Cargos with motors dispersed rigidly on their surface bind microtubules quickly, but are transported inefficiently. Cargos with freelydiffusing motors have both fast binding and efficient transport, although less efficient than clustered motors. These results point to a functional role for observed changes in motor organization on cargos, and suggest motor diffusivity as a control point for transport, either by modulation of adaptor proteins or changes in lipid composition.

No abstract