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Title:Dynamics of blue copper proteins
Author(s):Ehrenstein, David Henry
Doctoral Committee Chair(s):Frauenfelder, Hans
Department / Program:Physics
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Biochemistry
Biophysics, General
Abstract:Studies of small molecules binding to heme proteins have yielded a large amount of information about protein dynamics and conformational substates (CS) in proteins. However, heme proteins are very similar in their active site structures, and relatively little work exists on non-heme proteins which is directly comparable to the heme protein experiments. We have therefore studied the binding of NO to three blue copper proteins, a class of proteins which lack a heme group and have a different structural motif from that of heme proteins.
The proteins are azurin (Az), halocyanin (Ha), and nitrite reductase (NiR). NO binds reversibly to a type 1 copper active site in these proteins, and it can be photodissociated. We measured the kinetics of rebinding following photodissociation over a wide range of temperature (40-280 K) and time ($10\sp{-6}-10\sp2$ s) and compare the results with those of heme proteins. We find that the rebinding kinetics share many basic properties with heme protein kinetics. In all three proteins, at the lowest temperatures, rebinding is non-exponential in time, which in AzNO and HaNO is attributed to a Gaussian distribution of activation enthalpies of rebinding from a site close to the copper. Above 200 K, rebinding to Az and Ha is exponential in time and dependent upon NO concentration, implying that at these temperatures, fluctuations between CS are much faster than rebinding and that rebinding is bimolecular.
HaNO rebinding also exhibits two additional kinetic processes. The first one we model using an additional distribution of rebinding barriers having a non-Gaussian shape. We describe the second additional process using a relaxation model, which is based on a successful description of myoglobin-CO kinetics.
The flash photolysis kinetics of NiRNO display two distinct processes, one slow and one fast, with different physical bases. The slow process we attribute to NO rebinding, and we assign the fast process to intra-molecular electron transfer and describe it with a Gaussian distribution of enthalpy barriers. The fast process also directly demonstrates the phenomenon of fluctuational averaging, which has been previously observed in heme proteins. We show that the simplest model of this effect correctly predicts, within uncertainties, the average rate coefficient at high temperature.
Finally, we propose a model for the NO-bound copper coordination geometry and describe some implications of it. We conclude by summarizing the properties these blue copper proteins share with heme proteins and show that they not only confirm much of our understanding of protein dynamics but also exhibit a wider range of parameters than do heme proteins.
Issue Date:1995
Rights Information:Copyright 1995 Ehrenstein, David Henry
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9522106
OCLC Identifier:(UMI)AAI9522106

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