Effects of folding on metalloprotein active sites (protein foldingyrack formationycytochrome cyazurinyelectron transfer)

Experimental data for the unfolding of cytochrome c and azurin by guanidinium chloride (GuHCl) are used to construct free-energy diagrams for the folding of the oxidized and reduced proteins. With cytochrome c, the driving force for folding the reduced protein is larger than that for the oxidized form. Both the oxidized and the reduced folded forms of yeast cytochrome c are less stable than the corresponding states of the horse protein. Due to the covalent attachment of the heme and its fixed tetragonal coordination geometry, cytochrome c folding can be described by a two-state model. A thermodynamic cycle leads to an expression for the difference in self-exchange reorganization energies for the folded and unfolded proteins. The reorganization energy for electron exchange in the folded protein is approximately 0.5 eV smaller than that for a heme in aqueous solution. The finding that reduced azurin unfolds at lower GuHCl concentrations than the oxidized protein suggests that the coordination structure of copper is different in oxidized and reduced unfolded states: it is likely that the geometry of CuI in the unfolded protein is linear or trigonal, whereas CuII prefers to be tetragonal. The evidence indicates that protein folding lowers the azurin reorganization energy by roughly 1.7 eV relative to an aqueous Cu(1, 10-phenanthroline)2 reference system. The folding of a protein to its native three-dimensional structure is a spontaneous process, driven by the tendency of the peptide chain to assume the conformation of minimum free energy. As first clearly enunciated by Lumry and Eyring in 1954 (1, 2), the universal minimum for a given protein (i.e., for a specific amino acid sequence) may be reached at the expense of some local energy maximum. They further suggested that evolution has availed itself of this so-called rack phenomenon to create strain and distortion in prosthetic groups or coenzymes, thereby tuning the electronic properties by the mechanical force. This idea also led to a visualization of evolutionary fine tuning of active-site properties in protein superfamilies by small variations in amino acid sequences. The idea of conformationally induced strain in protein active sites was further developed both by Lumry himself (3) and by other authors. Vallee and Williams (4) stressed, in particular, how strain in the active site of the ground state of a catalytic metalloenzyme (e.g., a blue copper protein) can poise the metal ion for its reaction with substrate. The unique properties of blue copper were first described in 1960 (5), and they were attributed to a rack mechanism by one of us in 1964 (6). The first attempt to estimate the rack energy for blue copper, based on ligand-field considerations, was published in 1983 (7), and recently, Brill (8) has developed a model to calculate the mechanical energy associated with stress and strain and applied it to one specific blue protein, azurin. Interestingly, electronic structure calculations (9, 10) and spectroscopic experiments (9) have suggested that there is little if any strain on CuII in a blue copper site, but that the bonding of methionine sulfur to CuI is weakened by forced elongation in a folded cupredoxin. These findings raise the possibility that the main role of the rack is to shield the copper from water and other potential ligands (10). Two detailed reviews (11, 12), featuring rather disparate accounts of developments in the field, have been published in the last few years. In this communication we will show how the properties of redox metalloproteins are related to the energetics of protein folding. We will discuss cytochrome c and azurin as examples, because the crystal structures of the wild-type (13, 14) as well as of several mutant proteins (15, 16) are available; there is a wealth of spectroscopic, thermodynamic, and kinetic data to draw upon (11, 15); and the folding of these proteins is being studied experimentally in our laboratories (17–19). Rack Formation by Folding In Fig. 1 we give a thermodynamic cycle for the folding of a redox protein (17). If the reduction potentials of the folded and unfolded protein are different, then the folding free energies of the oxidized and reduced proteins will differ by a corresponding amount. In a high-potential metalloprotein, the redox center in the folded state generally has a higher potential than in the unfolded protein, so that the driving force for folding is higher for the reduced protein. This can be due to the native fold destabilizing the oxidized metal or stabilizing the reduced center, or a combination of both. This is the essence of the rack concept (1, 2). Blue copper proteins provide good examples, in which both effects are operating (11). If the difference in folding free energies for the oxidized and reduced protein (D(DGf) [ DGf,OX 2 DGf,RED) is sufficiently large, it may be possible to find conditions in which the oxidized protein is completely unfolded, whereas the reduced one is fully folded. This is the basis for electron-transferinitiated folding (17, 18), which has brought folding studies into a much shorter time regime ($ nanoseconds) compared with that conventionally used (millisecond in stopped-flow dilution experiments). Recent theoretical work (20, 21) suggests that this is a necessity for studies of the initial collapse to a compact denatured state. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y944246-4$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: phen, 1,10-phenanthroline; bpy, 2,29-bipyridine; GuHCl, guanidinium chloride; NHE, normal hydrogen electrode; Cyt c, cytochrome c. ¶To whom reprint requests should be addressed at: Beckman Institute 139–74, California Institute of Technology, Pasadena, CA 91125. e-mail: [email protected].