Proc. Nati. Acad. Sci. USA Vol. 91, pp. 11118-11122, November 1994 Biochemistry A cation binding motif stabilizes the compound I of (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 reacts with peroxide electron paramagnetic resonance and external nuclear double to form compound I, which contains an oxyferryl 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 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 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 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 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 , 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). Solvent was treated as a continuum with a dielec- methyl groups and several nearby residues. tric constant of e = 80 and the dielectric constant of the The location of the cation varied by <1 A among these protein was specified as E = 2 or e = 9. Ionic strength was set structures, indicating that a single well-defined cation binding at 100 mM. The relative contribution of individual groups to site is present. This site is composed of two structural the electrostatic potential was evaluated by removing partial elements: a type III reverse turn formed by residues 174-177 charges on specified atoms. For carbonyl charges, changes and an a-helix formed by residues 165-175. The framework were made in C and 0 atoms to maintain electroneutrality. for the binding site is provided by a standard type III turn (24), in which the backbone carbonyl oxygen atoms of RESULTS AND DISCUSSION His-175 (residue 2 of the turn) and Leu-177 (residue 4 of the turn) approach within 3.8 A of each other. This geometry Crystal structures of the Trp-191 -* Gly and Trp-191 -- Gln allows the partial negative charge on both carbonyl oxygen mutants were determined at 2.2-A resolution (Tables 1 and 2). atoms to interact with the bound cation (Fig. 2). Additionally, The CcP(MI,G191) mutation produced a cavity near the heme the type III turn occurs at the carboxyl terminus of a long that was readily observed in FO[CcP(MI,G191)] - FO[CcP- a-helix, allowing the bidentate cation binding site to interact (MI)] electron density maps, but all attempts to detect indole, favorably with the negative end ofthe helix macrodipole (25). purine, or benzene soaked into the cavity were unsuccessful. Reverse-turn structures of this type contribute to K+ Instead, buffer cations were detected within the cavity. binding by two other enzymes, dialkylglycine decarboxylase These included K+, Tris, and ammonium for the Trp-191 --+ (26) and subtilisin (27). In all three cases, the K+ binding Gly enzyme and K+ for the Trp-191 -3 Gln enzyme. The motif consists of a type I or type III turn that brings the presence of K+ is clearly shown by the large positive density backbone carbonyl ox gen atoms of its second and fourth in the IFO(K+) - FO(NHt)Iaic.,_ map (Fig. 1A). A bound Tris residues to within =4 A of each other. This geometry allows molecule is indicated by the large positive density in the same the respective oxygen atoms to contribute one equatorial and region, as shown by the IFO(Tris+) - Fc(NH )Iacwc map in Fig. 1B. Refined models of the cation binding cavity region of two tAt the resolution of the x-ray data, it is not possible to differentiate between a water molecule and an ammonium ion. The molecule in mutant enzymes are shown in Fig. 2. In the large cavity of the cation binding site is assumed to be an ammonium ion rather CcP(MI,G191), K+ assumes an octahedral coordination (Fig. than a water molecule, based on the tendency of this site to bind 2B) similar to that described for valinomycin (23). Two other monovalent cations. Table 1. Crystal data and diffraction statistics Cell dimensions, A Res, No. total observations/ Completeness, Rsymst Crystal a b c A I/O"* no. unique reflections % % Q191 105.2 74.2 45.2 2.2 3.4 71,833/16,458 87.1 5.5 G191, K+ 105.0 74.3 45.1 2.1 2.8 49,952/16,205 86.1 4.0 G191, NHW 105.0 74.2 45.1 2.1 2.5 65,268/20,136 94.8 4.4 G191, Tris+ 105.0 74.1 45.0 2.2 2.3 57,264/17,182 93.4 5.5 Res, resolution. *Average ratio of intensity (I) to a, for the highest resolution shell. tRSym = EIIobs - Iavgl/X lavg- Downloaded by guest on September 25, 2021 11120 Biochemistry: Miller et al. Proc. Natl. Acad. Sci. USA 91 (1994) A C A

177 BX

B AHis 175

.9.. 2<~~I K Tris + Gly 19\

HIS 175 r Leu 177 FIG. 2. Refined structures of CcP(MI,Q191) in potassium phos- FIG. 1. Dependence of electron density in the cavity created by phate (A) and of CcP(MI,G191) in potassium phosphate (B), Mes/ the Trp-191 -. Gly mutation upon the cation composition of the ammonium chloride buffer (C), or Mes/Tris buffer (D). Tetrahedra mother liquor. (A) Difference electron density map for CcP- indicate potassium ions, the ammonium ion, and bound water (MI,G191) crystallized in potassium phosphate buffer or in Mes/ molecules. Dashed lines are drawn between the potassium ions and ammonium chloride buffer IFo(K+) - F0(NHW)Iaicj,. The map is its ligands inA and B and indicate important hydrogen bonds in C and contoured at +5 of and is superimposed upon the refined model of D. CcP(MI,G191) crystallized in potassium phosphate buffer. (B) Dif- ference electron density map for CcP(MI,G191) crystallized in Mes/ ofcharged atoms. Contributions to this electrostatic potential Tris buffer or in Mes/ammonium chloride buffer: IFO(Tris+) - arise from the backbone carbonyl oxygen atoms (-6.5 kcal Fc(NH+)Iacaic. FC(NH ) was calculated with all water molecules and per mol per electron), the macrodipole from the a-helix the ammonium ion deleted from the cavity. The map is contoured at formed by residues 165-175 (-1.5 kcal per mol per electron), 5 a and is superimposed upon the refined model of CcP(MI,G191) the heme propionates (-6.4 kcal per mol per electron), and crystallized in Tris buffer. Asp-235 (-7.9 kcal per mol per electron). Although the point one axial ligand to the K+ ion. Such a binding site will occur of strongest electrostatic potential is found at the K+ binding in any type I or type III turn where dihedral angles 04 - 60 site, the entire indole ring of Trp-191 is surrounded by a and q4 =140. The similarity of this structural feature in all negative electrostatic potential in excess of -27 kcal per mol three ofthe known K+ binding sites suggests that it is awidely per electron (Fig. 3). occurring motif for alkali cation-binding enzymes. On the These electrostatic potential calculations provide strong other hand, the interaction ofthe cation with a helix dipole is evidence that the Trp-191 radical is a cation. The pKa values not the K+ sites of forthe Asp-235 carboxylate and the indolyl radical are similar apparently required; binding dialkylgly- in aqueous medium (28), but the electrostatic potential will cine decarboxylase or subtilisin have no such feature. A helix shift the pKa of the Trp-191 radical substantially upward in dipole does contribute to the Na+ binding site of dialkylgly- CcP(MI). Because the negative potential arises from several cine decarboxylase, however. moieties surrounding Trp-191, no single peak of negative By unmasking a cation binding site via mutagenesis, we potential is created at the indole ring nitrogen atom (NE1) of have shown that a substantial negative electrostatic potential Trp-191 by its interaction with Asp-235. In fact, the strong exists at the site occupied by the indole ring ofTrp-191 in the negative potential encompassing the indole ring (Fig. 3) may parent enzyme. This potential will stabilize an indolyl cation promote a partial or complete delocalization of the positive radical produced by oxidation ofthe indole ring relative to the charge onto the carbon atoms ofthe six-membered ring. This neutral indole ring ofthe resting enzyme, thus decreasing the phenomenon may be responsible for the contradictory con- midpoint potential for Trp-191 oxidation. Moreover, the clusions reached by others regarding the protonation state of preferential binding ofcations over electrically neutral planar the Trp-191 radical (11, 12). molecules in the cavity ofthe CcP(MI,G191) mutant leads us Several lines of experimental evidence provide qualitative to conclude that an indole moiety will occupy this site only support for our electrostatic calculations. Mutagenesis has when tethered by covalent bonds to the protein backbone. shown that when Asp-235 is replaced by Asn, the Trp 191 side To evaluate the importance of the cation binding site in chain rotates about the bond between the (- and -y-carbon stabilizing the compound I radical, it is necessary to consider atoms. This places NE1 of the indole ring within hydrogen the approximate magnitude of the electrostatic potential in bonding distance of the backbone carbonyl of Leu-177 (19). this region ofthe enzyme. The electrostatic potential field for The calculations indicate that the strong negative potential CcP(MI,G191) was estimated with the aid of the program near the Leu-177 carbonyl makes this the best alternative DELPHI (Biosym Technologies). Our computational simula- hydrogen bond acceptor available to Trp-191. Apparently, tion confirms the presence of a region of strong negative the change in orientation of the indole ring and the loss of electrostatic potential near residue 191 (Fig. 3). At the center negative charge from the Asp-235 side chain eliminate the ofthe K+ binding site, the calculated potential is -29 kcal per facile oxidation of Trp-191 (16, 29). Moreover, esterification mol per electron, which represents the strongest region of of both heme propionates decreases the enzyme activity by negative potential within the enzyme molecule, excluding the 99.5% and decreases the stability of the compound I radical, electrostatic potential enclosed by the van der Waals surfaces without significantly altering the rate of reaction of the Downloaded by guest on September 25, 2021 Biochemistry: Miller et al. Proc. NatL. Acad. Sci. USA 91 (1994) 11121

FIG. 3. Calculated electrostatic potential near the cation binding site ofCcP(MI,G191). After least squares superposition ofmodel structures for CcP(MI,G191) with the CcP(MI) parent, electrostatic potential contours of CcP(MI,G191) were calculated in the plane ofCcP(MI)-Trp-191 indole ring. The contours are labeled to indicate the calculated free energy change (in kcal/mol) for placing a positive charge at a given point within the electrostatic field. The K+ ion is represented as a solid sphere near Trp-191. The peptide backbone of residues 175-177 and the side chain of Asp-235 from the CcP(MI,G191) model are also shown. Van der Waals surfaces for K+, the carbonyl oxygen atoms of His-175 and Leu-177, and the carboxylate oxygen atoms of Asp-235 are represented as dotted spherical surfaces. enzyme with peroxide (30, 31). Since the steady-state activity The symbol R in Eq. 2 represents an endogenous donor that is sensitive to the equilibrium between the oxyferryl heme is not Trp-191 (e.g., a Tyr residue). The rate constant for the and the Trp-191 radical (8), these effects are consistent with reaction of the porphyrin radical with this internal reductant a stabilizing influence of the negatively charged propionate is quite fast (k2 = 50 sec1 at pH 6 and 250C) and the half-life groups on the Trp-191 radical. of the radical is =14 msec (2). While it is clear that the experimental results are in good If we make the additional assumption that the porphyrin qualitative agreement with the electrostatic potential calcu- radical of native CcP has the same rate of endogenous lations, it is more difficult to assess the quantitative accuracy reduction as the porphyrin radical formed by the Trp-191 -+ ofthe calculated potentials. As noted by Aqvist et al. (32), the Phe mutant, the rate of endogenous reduction of the com- magnitude of the electrostatic potential depends upon the pound I radical can be calculated on the basis of Eqs. 1 and local dielectric, a quantity that cannot be easily determined. 2. The rate constant for internal reduction of the Trp-191 We can estimate the magnitude ofthe potential by calculating radical (kobs) will be the product ofthe fraction ofthe enzyme the minimum shift in midpoint potential for Trp-191 oxidation that has a radical on the porphyrin and the rate constant k2. that is consistent with the stability ofthe compound I radical. The fraction of enzyme that has a porphyrin radical can be This estimate can be made by comparing the rate of com- determined from K., since pound I decay in native CcP (5) with the rate of decay of the [Por'l porphyrin radical produced when the Trp-191 -* Phe enzyme Keq =_[ - [por+'1 reacts with peroxide (2). The calculation requires two as- eq[Trp-191+ l ET - [por+]'l sumptions that are fairly straightforward. [por+ l Keq First, it is assumed that when compound I is formed, the then p [31 radical at Trp-191 (Trp-191+ ) is in equilibrium with a por- ET (Keq+1) phyrin cation radical (pore') ofthe oxyferryl heme, as in Eq. 1. where ET is the total concentration of oxidized CcP- (MI,F191). The observed rate constant for internal reduction CcP(IV=O, por, Trp-191+") .CcP(IV=O, por+,', Trp-191) of Trp-191 is, therefore, given by [11] = k2 +) [4] Rapid equilibrium between the two radical types seems kobs (Keq(K + 1) inescapable, since the indole ring of Trp-191 is in van der Waals contact with the heme (13). This equilibrium must Since kobs = 10-4sec-1 (5) and Keq <« 1, strongly favor Trp-191 oxidation, however, since no porphy- rin radical has ever been observed in native CcP (i.e., Keq << kobs 10-4 sec1 - =2 xl10-. [51 1). Keq- k2 50 sec1 A transient porphyrin radical can be observed when Trp- 191 is replaced with Phe(2), but it is rapidly quenched by one If Kq = 2 x 10-6, then AG = -7.8 kcal/mol (or E' = 0.355 or more endogenous reductants on the enzyme, as shown in V) for the oxidation ofTrp-191 by the porphyrin radical. This Eq. 2. value is much greater than the standard oxidation potentials of the reacting species predict. The midpoint potential for CcP(IV=O, por+,', R) CcP(IV=O, por, R@) [2] oxidation of the oxyferryl porphyrin of horseradish peroxi- Downloaded by guest on September 25, 2021 11122 Biochemistry: Miller et al. Proc. Natl. Acad. Sci. USA 91 (1994)

dase is E' = 1 V at pH 6.0 (33), and the midpoint potential 7. Hahm, S., Geren, L., Durham, B. & Millet, F. (1993) J. Am. for oxidation of Trp in solution is in the range Eo = 0.99 - Chem. Soc. 115, 3372-3373. 1.18 V at pH 6.0 (15). By taking the lowest value for Trp 8. Hahm, S., Miller, M. A., Geren, L., Kraut, J., Durham, B. & Millet, F. (1994) Biochemistry 33, 1473-1480. oxidation, the equilibrium constant for this reaction should be 9. Hoffman, B. M., Roberts, J. E., Kang, C. H. &Margoliash, E. near 1, or E, = 0, implying that CcP stabilizes the Trp-191 (1981) J. Biol. Chem. 256, 6556-6564. radical by at least 7.8 kcal/mol (or 355 mV) relative to a Trp 10. Hoffman, B. M., Roberts, J. E., Brown, T. G., Kang, C. H. & radical in solution. This is substantially less than the calcu- Margoliash, E. (1979) Proc. Nati. Acad. Sci. USA 76, 6132- lated free energy change AG = -27 kcal/mol for cation 6136. binding in the Trp-191 site. Moreover, the electrostatic 11. Houseman, A. L., Doan, P. E., Goodin, D. B. & Hoffman, potential at Trp-191 is sufficient to account for the stability of B. M. (1993) Biochemistry 32, 4430-4443. the indole radical even if a much larger effective dielectric 12. Krauss, M. & Garmer, D. R. (1993) J. Phys. Chem. 97, constant is assumed. For example, ifthe value ofthe effective 831-836. 13. Finzel, B. C., Poulos, T. L. & Kraut, J. (1984) J. Biol. Chem. dielectric eeff = 9, as suggested by Aqvist et al. (32) for 259, 13027-13036. barnase, a value ofAG = -7.5 kcal/mol is obtained for cation 14. Kadish, K. M., Morrison, M. M., Constant, L. A., Dickens, binding in this site. L. & Davis, D. G. (1976) J. Am. Chem. Soc. 98, 8387-8390. 15. DeFelippis, M. R., Murthy, C. P., Faraggi, M. & Klapper, Note. While this manuscript was in preparation, it was reported that M. H. (1989) Biochemistry 28, 4847-4853. several imidazole derivatives bind weakly in the cavity created by a 16. Miller, M. A., Bandyopadhyay, D., Mauro, J. M., Traylor, Trp-191 -) Gly mutant of CcP (34). The pH dependence of binding T. G. & Kraut, J. (1992) Biochemistry 31, 2789-2797. indicated that the ligands were imidazolium cations (34). It is 17. Eriksson, A. E., Baase, W. A., Zhang, X. J., Heinz, D. W., important to stress that there is no conflict between the very strong Blaber, M. & Matthews, B. W. (1992) Science 255, 178-183. cation binding predicted by the present work and the very weak 18. Fishel, L. A., Villafranca, J. E., Mauro, J. M. & Kraut, J. binding of imidazolium and its derivatives to a similar Trp-191 -* Gly (1987) Biochemistry 26, 351-360. mutant of CcP reported elsewhere (34). We have shown that the 19. Wang, J., Mauro, J. M., Edwards, S. L., Oatley, S. J., Fishel, cation binding site created by the Trp-191 -- Gly mutation is not near L. A., Ashford, V. A., Xuong, N.-h. & Kraut, J. (1990) Bio- the carboxylate of Asp-235 as claimed (34) but is between the chemistry 29, 7160-7173. carbonyl oxygen atoms of residues 175 and 177. From the low 20. Xuong, N.-H., Nielsen, C. P., Hamlin, R. & Anderson, D. H. crystallographic B factor (B = 7.6) of the "water" molecule in the (1985) J. Appl. Crystallogr. 18, 342-350. cation binding site (Wat 400 in ref. 34), we infer that a buffer cation 21. Tronrud, D. E., Ten Eyck, L. F. & Matthews, B. W. (1987) occupies this site in the ligand-free structure. The very weak binding Acta Crystallogr. Sect. A 43, 489-501. constant reported for imidazolium in this cavity simply indicates that 22. Collins, J. R. & Loew, G. H. (1992) Int. J. Quantum Chem. 19, the imidazolium ion competes poorly for the site occupied by Na+ 87-107. [which was present at 30 mM in the soaking buffer used by Fitzgerald 23. Neupert-Laves, K. & Dobler, M. (1975) Helv. Chim. Acta 58, et al. (34)]. 432-437. 24. Chou, P. Y. & Fasman, G. D. (1977)J. Mol. Biol. 115, 135-175. This paper is dedicated to the memory of our colleague, Teddy G. 25. Hol, W. M. G. (1985) Prog. Biophys. Mol. Biol. 45, 149-195. Traylor, whose passion for heme chemistry inspired much of our 26. Toney, M. D., Hohenester, E., Cowan, S. W. & Jansonius, work. We thank Dr. Dzung Nguyen of Biosym Technologies for J. N. (1993) Science 261, 756-759. assistance with the DELPHI software, Dr. J. Jansonius for providing 27. McPhalen, C. A. & James, M. N. G. (1988) Biochemistry 27, the coordinates for dialkylglycine decarboxylase, and Drs. J. Mat- 6582-6598. thew Mauro, Andrew Shaw, Michael Sawaya, Huguette Pelletier, 28. Posener, M. L., Adams, G. E., Wardman, P. & Cundall, R. B. and James Terner for helpful discussions. Support was provided by (1976) J. Chem. Soc. Faraday Trans. 72, 2231-2239. National Science Foundation Grant MCB 9119292. 29. Fishel, L. A., Farnum, M. F., Mauro, J. M., Miller, M. A., Kraut, J., Liu, Y., Tan, X. & Scholes, C. P. (1991) Biochem- 1. Mauro, J. M., Fishel, L. A., Hazzard, J. T., Meyer, T. E., istry 30, 1986-1996. Tollin, G., Cusonovich, M. A. & Kraut, J. (1988) Biochemistry 30. Asakura, T. & Yonetani, T. (1969) J. Biol. Chem. 244, 4573- 27, 6243-6256. 4579. 2. Erman, J. E., Vitello, L. B., Mauro, J. M. & Kraut, J. (1989) 31. Dowe, R. J. & Erman, J. E. (1982) J. Biol. Chem. 257, 2403- Biochemistry 28, 7992-7995. 2405. 3. Scholes, C. P., Liu, Y., Fishel, L. A., Farnum, M. F., Mauro, 32. Aqvist, J., Luecke, H., Quiocho, F. A. & Warshel, A. (1991) J. M. & Kraut, J. (1989) Isr. J. Chem. 29, 85-92. Proc. Nati. Acad. Sci. USA 88, 2026-2030. 4. Sivaraja, M., Goodin, D. B., Hoffman, B. M. & Smith, M. B. 33. Dunford, H. B. (1991) in in Chemistry and Biol- (1989) Science 245, 738-740. ogy, eds. Everse, J., Everse, K. E. & Grisham, M. B. (CRC, 5. Erman, J. E. & Yonetani, T. (1975) Biochim. Biophys. Acta Boca Raton, FL), Vol. 2, pp. 1-24. 393, 350-357. 34. Fitzgerald, M. M., Churchill, M. J., McRee, D. E. & Goodin, 6. Pelletier, H. & Kraut, J. (1992) Science 258, 1748-1755. D. B. (1994) Biochemistry 33, 3807-3818. Downloaded by guest on September 25, 2021