Electron transfer within complexes of cytochrome c (Cc) and cytochrome c peroxidase (CcP) was studied to determine whether the reactions are gated by fluctuations in configuration. Electron transfer in the physiological complex of yeast Cc (yCc) and CcP was studied using the Ru-39-Cc derivative, in which the H39C/C102T variant of yeast iso-1-cytochrome c is labeled at the single cysteine residue on the back surface with trisbipyridylruthenium(II). Laser excitation of the 1:1 Ru-39-Cc-CcP compound I complex at low ionic strength results in rapid electron transfer from RuII to heme c FeIII, followed by electron transfer from heme c FeII to the Trp-191 indolyl radical cation with a rate constant keta of 2 x 10(6) s-1 at 20 degrees C. keta is not changed by increasing the viscosity up to 40 cP with glycerol and is independent of temperature. These results suggest that this reaction is not gated by fluctuations in the configuration of the complex, but may represent the elementary electron transfer step. The value of keta is consistent with the efficient pathway for electron transfer in the crystalline yCc-CcP complex, which has a distance of 16 A between the edge of heme c and the Trp-191 indole [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. Electron transfer in the complex of horse Cc (hCc) and CcP was examined using Ru-27-Cc, in which hCc is labeled with trisbipyridylruthenium(II) at Lys-27. Laser excitation of the Ru-27-Cc-CcP complex results in electron transfer from RuII to heme c FeII with a rate constant k1 of 2.3 x 10(7) s-1, followed by oxidation of the Trp-191 indole to a radical cation by RuIII with a rate constant k3 of 7 x 10(6) s-1. The cycle is completed by electron transfer from heme c FeII to the Trp-191 radical cation with a rate constant k4 of 6.1 x 10(4) s-1. The rate constant k4 decreases to 3.4 x 10(3) s-1 as the viscosity is increased to 84 cP, but the rate constants k1 and k3 remain the same. The results are consistent with a gating mechanism in which the Ru-27-Cc-CcP complex undergoes fluctuations between a major state A with the configuration of the hCc-CcP crystalline complex and a minor state B with the configuration of the yCc-CcP complex. The hCc-CcP complex, state A, has an inefficient pathway for electron transfer from heme c to the Trp-191 indolyl radical cation with a distance of 20.5 A and a predicted value of 5 x 10(2) s-1 for k4A. The observed rate constant k4 is thus gated by the rate constant ka for conversion of state A to state B, where the rate of electron transfer k4B is expected to be 2 x 10(6) s-1. The temperature dependence of k4 provides activation parameters that are consistent with the proposed gating mechanism. These studies provide evidence that configurational gating does not control electron transfer in the physiological yCc-CcP complex, but is required in the nonphysiological hCc-CcP complex.

Given the importance of protein complexes as therapeutic targets, it is necessary to understand the physical chemistry of these interactions under the crowded conditions that exist in cells. We have used sedimentation equilibrium to quantify the enhancement of the reversible homodimerization of ␣-chymotrypsin by high concentrations of the osmolytes glucose, sucrose, and raffinose. In an attempt to rationalize the osmolytemediated stabilization of the ␣-chymotrypsin homodimer, we have used models based on binding interactions (transfer-free energy analysis) and steric interactions (excluded volume theory) to predict the stabilization. Although transfer-free energy analysis predicts reasonably well the relatively small stabilization observed for complex formation between cytochrome c and cytochrome c peroxidase, as well as that between bobtail quail lysozyme and a monoclonal Fab fragment, it underestimates the sugar-mediated stabilization of the ␣-chymotrypsin dimer. Although predictions based on excluded volume theory overestimate the stabilization, it would seem that a major determinant in the observed stabilization of the ␣-chymotrypsin homodimer is the thermodynamic nonideality arising from molecular crowding by the three small sugars.

The cytoplasm contains high concentrations of cosolutes. These cosolutes include macromolecules and small organic molecules called osmolytes. However, most biophysical studies of proteins are conducted in dilute solutions. Two broad classes of models have been used to describe the interaction between osmolytes and proteins. One class focuses on excluded volume effects, while the other focuses on binding between the protein and the osmolyte. To better understand protein-smolyte interactions, we have conducted sedimentation equilibrium analytical ultracentrifugation experiments using ferricytochrome c as a model protein.From these experiments, we determined the second virial coefficients for a series of osmolytes. We have interpreted the second virial coefficient as a measure of both excluded volume and protein-osmolyte binding. We conclude that simple models are not sufficient to understand the interactions between osmolytes and proteins. Keywords: Excluded volume; second virial coefficient; cytochrome c; osmolytes; analytical ultracentrifugationThe cellular medium in which proteins must function contains high concentrations of cosolutes. The cosolutes include proteins, nucleic acids, and osmolytes, low molecular weight organic molecules with no net charge. Some organisms change their osmolyte composition in response to environmental stresses. For example, cartilaginous marine fish use trimethylamine N-oxide (TMAO) to counteract high concentrations of urea (Yancey et al. 1982). Here, we present data on the interaction between iso-1-ferricytochrome c and osmolytes.Contrary to cellular conditions, most biophysical studies are conducted in dilute solutions (i.e., in the absence of cosolutes). Therefore, data from these studies may bear little resemblance to the way proteins behave in vivo (Zimmerman and Minton 1993).Many osmolytes stabilize proteins in vivo and in vitro (Hofmeister 1888;Singer and Lindquist 1998;Saunders et al. 2000). However, the stabilization mechanism is poorly understood. In general, two classes of models are used to explain the effects of osmolytes on protein stability (Saunders et al. 2000 and references therein). The first class focuses on the binding between osmolytes and proteins. The other class focuses on excluded volume effects arising from the increased steric repulsions between osmolytes and the protein. The binding models claim that osmolyte-induced stability increases arise from preferential binding of the osmolyte to the native state. The excluded volume models focus on the fact that osmolytes limit the conformational freedom of proteins by driving them to their most compact state, the native state. The decrease in conformational freedom arises from steric repulsions between the protein and the osmolyte.The actual mechanism must be a combination of the two classes, and models based on this combination lead to valuable insight. The fact that osmolytes take up space in solution cannot be denied. The resulting steric repulsion is shown by osmolyte-induced stabilization of the A...

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