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Cytochrome C Peroxidase (Protein Crystaflography/Site-Drected Mutagenesis/Electrostatic Potential) MARK A

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Cytochrome C Peroxidase (Protein Crystaflography/Site-Drected Mutagenesis/Electrostatic Potential) MARK A Proc. Nati. Acad. Sci. USA Vol. 91, pp. 11118-11122, November 1994 Biochemistry A cation binding motif stabilizes the compound I radical of cytochrome c peroxidase (protein crystaflography/site-drected mutagenesis/electrostatic potential) MARK A. MILLER, GYE WON HAN, AND JOSEPH KRAUT Department of Chemistry, University of California at San Diego, La Jolla, CA 92093-0317 Contributed by Joseph Kraut, May 26, 1994 ABSTRACT Cytochrome c peroxidase reacts with peroxide electron paramagnetic resonance and external nuclear double to form compound I, which contains an oxyferryl heme and an resonance spectroscopy (ENDOR) spectra of the radical led indolyl radical at Trp-191. The indolyl free radical has a half-life to its incorrect initial assignment as a sulfur radical (9, 10). of several hours at room temperature, and this remarkable Subsequent mutagenesis experiments combined with EN- stabiity Is essential for the catalytic function of cytochrome c DOR yielded the correct assignment ofTrp-191 as the radical peroxidase. To probe the protein environment that stabile the site (1-3). The location of the compound I radical at a Trp compound I radical, we used site-directed mutagenesis to re- residue was later confirmed by ENDOR experiments that place Trp-191 with Gly or Gin. Crystal structures of these used CcP specifically deuterated at Met or Trp residues (4). mutants revealed a monovalent cation binding site in the cavity The assignment of the compound I radical to Trp-191 formerly occupied by the side chain of Trp-191. Comparison of makes it possible to examine the relationship between the this site with those found in other known cation binding enzymes local protein structure and the unique properties of this shows that the Trp-191 side chain resides in a consensus K+ radical. One issue that must be considered is whether the binding site. Electrostatic potential calculations indiate that the compound I radical is a cationic (TrpH+) or a neutral species cation binding site is created by partil negative charges at the (Trp). Spectroscopy alone has failed to unambiguously dis- backbone carbonyl oxygen atoms of residues 175 and 177, the tinguish between these two possibilities (11, 12). A second carboxyl end of a long a-helix (residues 165-175), the heme important issue to be addressed is how the remarkable proplonates, and the carboxylate side chain of Asp-235. These stability of the Trp-191 radical (5) can be reconciled with its features create a negative potential that envelops the side chain location in van der Waals contact with the porphyrin and <8 ofTrp-191; the caculated free energy change for cation binding A from nearby Tyr residues (13). A normal Trp radical would in this site is -27 kcal/mol (1 cal = 4.184J). This is more than be rapidly quenched in this environment (14, 15). The sta- suffiient to account for the stability of the Trp-191 radical, bility of the compound I radical must, therefore, rely upon a which our estimates suggest is stabilized by 7.8 kcal/mol relative substantial decrease in the midpoint potential for Trp-191 to a Trp radical in solution. oxidation or a substantial increase in the midpoint potential for the surrounding residues. The former possibility is clearly Cytochrome c peroxidase (CcP) catalyzes the two-electron indicated by the observation that the rapid oxidation of reduction of peroxide to water, with ferrocytochrome c serv- Trp-191 by dioxygen bound to CcP(II) is eliminated when the ing as the electron donor. When a hydrogen peroxide molecule environment of Trp-191 is perturbed (16). reacts initially with the ferric heme of CcP, the two oxidizing To probe the environment of Trp-191, we have employed equivalents of peroxide are retained by the enzyme as an site-directed mutagenesis to replace the side chain of Trp-191 oxyferryl (Fe+Y4O) center and an indolyl radical at Trp-191 with Gly or Gln, thereby creating an internal cavity in the (1-4). This oxidized state, designated compound I, is stable for cloned CcP expressed in Escherichia coli [CcP(MI)J. The several hours in the absence of reductant (5) but rapidly mutant enzymes were subsequently crystallized in the pres- returns to the ferric state via sequential reaction with two ence of small molecules that could bind within the cavity, and molecules of ferrocytochrome c. One remarkable aspect of structures ofthe enzyme-ligand complexes were determined. this electron transfer reaction is that the heme edges of the The strategy is similar to that employed by Matthews and reacting partners remain separated by no less than 18 A in the coworkers (17), who found that internal cavities created within complex (6). T4 lysozyme could bind small organic molecules. The Trp-191 residue of CcP is critical for this long-distance In the present report, we demonstrate that cations, includ- electron transfer reaction. One oxidizing equivalent ofperox- ing K+, Tris, and ammonium, bind in the cavity created when ide is retained initially as a stable radical at Trp-191 by Trp-191 is replaced with Gly or Gln. Moreover, calculations ferrocytochrome c. In addition, facile oxidation of Trp-191 is reveal that the cation binding site is at the center of a large required for rapid electron transfer from cytochrome c to the region of negative electrostatic potential that is of sufficient compound I oxyferryl center (1, 3, 7, 8). Thus, the Trp-191 magnitude to account for the stability of the compound I radical constitutes an electron "gate" that allows the con- radical.* trolled reduction of peroxide, a two-electron oxidant, by cytochrome c, a one-electron reductant. Elucidation of the MATERIALS AND METHODS interaction between Trp-191 and its local environment is, therefore, critical to understanding this gated electron transfer Preparation of Enzymes. Techniques employed for muta- reaction of CcP. genesis of CcP(MI), expression, and purification of the The CcP-compound I radical has unique properties that made its identification by spectroscopy difficult. The unusual Abbreviations: CcP, cytochrome c peroxidase; CcP(MI), the cloned CcP expressed in Escherichia coli. *The atomic coordinates and structure factors have been deposited The publication costs of this article were defrayed in part by page charge in the Protein Data Bank, Chemistry Department, Brookhaven payment. This article must therefore be hereby marked "advertisement" National Laboratory, Upton, NY 11973 (references 1CPD, 1CPE, in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1CPF, and 1CPG). This information is not embargoed. 11118 Downloaded by guest on September 25, 2021 Biochemistry: Miller et al. Proc. Natl. Acad. Sci. USA 91 (1994) 11119 recombinant CcP(MI) have been described (18). To examine Table 2. Refinement statistics the cation binding affinity of CcP(MI,G191), this mutant rms deviations from ideal R enzyme was purified and crystallized for diffraction experi- ments in the absence of K+. After fractionation on a Seph- Bond, Angle, Torsion, Planes, Resolution factor, adex G-75 column, the crude enzyme was separated into Model A deg deg A range, A % holoenzyme and apoenzyme fractions. Apoenzyme was con- Q191 0.009 3.06 16.9 0.005 20-2.2 16.0 verted to holoenzyme by standard procedures (18), after G191, K+ 0.009 3.03 16.7 0.005 20-2.2 16.6 exhaustive dialysis against either Mes/NH4Cl/NH4OH or G191, NHW 0.012 2.72 15.7 0.007 20-2.2 16.4 Mes/Tris buffers of the appropriate ionic strength and pH. G191, Tris+ 0.011 2.61 16.1 0.007 20-2.2 16.1 Subsequent purification and crystallization ofthe ammonium R = X IFobs - Fcall/I Fobs. deg, Degrees. and Tris forms of CcP(MI,G191) were carried out in one of the two buffer systems noted above. ligands are contributed by backbone carbonyl oxygen atoms, X-Ray Crystallography. Crystallization ofthe CcP mutants and three water molecules are also coordinated to the K+ ion. was performed according to Wang et al. (19). Crystals of Due to steric constraints of the enzyme, the sixth coordina- CcP(MI,G191) with K+, ammonium, or Tris cations bound in tion site remains unoccupied. In the smaller cavity of CcP- the cavity and the crystals of CcP(MI,Q191) with K+ bound (MI,Q191), coordination of K+ is tetrahedral (Fig. 2A). were all isomorphous with the CcP(MI) parent P212121 crys- Ligands for K+ include the backbone carbonyl oxygen atoms tals. Cell dimensions of CcP(MI) were a = 104.9 A, b = 74.2 of residues 175 and 177, the side-chain carbonyl of Gln-191, A, and c = 45.5 A. X-ray data were collected on a multiwire and a water molecule. The presumed ammonium iont bound area detector (20) with monochromatic CuKa radiation. Es- to CcP(MI,G191) also exhibits tetrahedral geometry (Fig. sential statistics are summarized in Table 1. Model structures 2C), forming hydrogen bonds with two water molecules in were refined using the TNT restrained least squares program addition to the backbone carbonyls of residues 175 and 177. (21). Refinement statistics are summarized in Table 2. A more complicated interaction is seen when the Tris cation lectrostatic Potential Calculations. Electrostatic potential binds to CcP(MI,G191) (Fig. 2D). Although the ammonium calculations were performed with DELPHI software (Biosym moiety is located in the same position as K+, where it forms Technologies, San Diego). Atoms were assigned partial a hydrogen bond with the carbonyl oxygen ofHis-175, one of charges according to program defaults, with His-181 speci- the hydroxymethyl groups forms a hydrogen bond with the fied as an imidazolium ion. Partial charges on the heme and carbonyl oxygen ofLeu-177. An extensive hydrogen bonding proximal His-175 ligand were assigned from INDO calcula- network is also formed between the remaining hydroxy- tions (22).
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