Conformational substates in azurin.

Abstract

Azurin is a small blue copper protein in the electron transfer chain of denitrifying bacteria. It forms a photolabile complex with nitric oxide (NO) at low temperatures. We studied the temperature dependence of the ligand binding equilibrium and the kinetics of the association reaction after photodissociation over a wide range of temperature (80-280 K) and time (10-6-102 s). The nonexponential rebinding below 200 K is independent of the NO concentration and is interpreted as internal recombination. The rebinding can be modeled with the Arrhenius law by using a single preexponential factor of 6.3 x 108 s-' and a Gaussian distribution of enthalpy barriers centered at 23 kJ/mol with a width of 11 kJ/mol. Above 200 K, a slower, exponential rebinding process appears. The dependence of the kinetics on the NO concentration characterizes this reaction as bimolecular rebinding. The binding kinetics of NO to azurin show impressive analogies to the binding of carbon monoxide to myoglobin. We conclude that conformational substates occur not only in heme proteins but also in proteins with different active sites and secondary structures.Experiments measuring the binding of small ligands to heme proteins have contributed considerably to our understanding of the relation between the structure, dynamics, and function of proteins (1, 2). Sperm whale myoglobin (Mb) has served as a model system in these studies. Mb, a small, globular protein of 17.8 kDa, consists of 153 amino acids folded into a globular structure with eight a-helices. Diatomic ligands (02, CO) bind at the heme iron. The rebinding after photodissociation is nonexponential in time at low temperatures. An important concept results from the kinetic studies-namely, that proteins do not exist in a unique, well-defined structure, but in a large number of slightly different structures, the conformational substates (CS). Evidence for CS also comes from analysis of the Debye-Waller factor in protein crystals by x-ray diffraction (3, 4) and from the inhomogeneity of spectral lines (5, 6). The CS appear to be grouped into different tiers with distinctly different free energy barriers between the substates, leading to a hierarchical model of the conformational energy landscape in MbCO (7,8).We believe that these concepts apply not only to MbCO but to proteins in general. Strong covalent bonds establish the primary sequence, while only relatively weak forces like hydrogen bonds and hydrophobic effects determine the folding into the three-dimensional structure. Consequently, the positions of the amino acids are not unique, and competition among neighboring amino acids for the energetically optimal configuration arises from the dense packing. These two effects, disorder and frustration, lead to a complex conformational energy landscape with many local minima, the CS. The hierarchical arrangement of the CS may reflect the hierarchy of structural features in proteins.To study the generality of these concepts, we chose azurin (Az) (from Pseudomonas aeruginosa), a protein of ...

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