KRAUT RESEARCH GROUP ABSTRACTS

CONTENTS:

CYTOCHROME C PEROXIDASE

  • Crystal structure of a complex between electron transfer partners, cytochrome c peroxidase and
    cytochrome c

  • 2.2 angstrom structure of oxy-peroxidase as a model for the transient enzyme:peroxide complex

  • Interaction domain for the reaction of cytochrome c with the radical and the oxyferryl heme in cytochrome c peroxidase compound I

  • A cation binding motif stabilizes the compound I radical of cytochrome c peroxidase

  • Regulation of interprotein electron transfer by Trp 191 of cytochrome c peroxidase

  • Identifying the physiological electron transfer site of cytochrome c peroxidase by structure-based engineering

    DNA POLYMERASE BETA

  • Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism

  • Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP

    DIHYDROFOLATE REDUCTASE

  • Isomorphous crystal structures of Escherichia coli dihydrofolate reductase complexed with folate, deazafolate, and 5,10-dideazatetrahydrofolate: mechanistic implications

  • Crystal structures of escherichia coli dihydrofolate reductase complexed with 5-formyltetrahydrofolate (folinic acid) in two space groups - evidence for enolization of pteridine O4

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    Crystal structure of a complex between electron transfer partners, cytochrome c peroxidase and cytochrome c.

    Pelletier, H., and J. Kraut

    Science, Vol. 258, pp. 1748-1755, (1992).

    The crystal structure of a 1:1 complex between yeast cytochrome c peroxidase and yeast iso-1-cytochrome c was determined at 2.3 A resolution. This structure reveals a possible electron transfer pathway unlike any previously proposed for this extensively studied redox pair. The shortest straight line between the two hemes closely follows the peroxidase backbone chain of residues Ala194, Ala193, Gly192, and finally Trp191, the indole ring of which is perpendicular to, and in van der Waals contact with, the peroxidase heme. The crystal structure at 2.8 A of a complex between yeast cytochrome c peroxidase and horse heart cytochrome c was also determined. Although crystals of the two complexes (one with cytochrome c from yeast and the other with cytochrome c from horse) grew under very different conditions and belong to different space groups, the two complex structures are closely similar, suggesting that cytochrome c interacts with its redox partners in a highly specific manner.

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    2.2 angstrom structure of oxy-peroxidase as a model for the transient enzyme:peroxide complex

    Miller, M.A., A. Shaw, J. Kraut

    Nature Structural Biology, Vol. 1, pp. 523-533, (1994).

    The Fe+3-OOH complex of peroxidases has a very short half life, and its structure cannot be determined by conventional methods. The Fe+2-O2 complex provides a useful structural model for this intermediate, as it differs by only one electron and one proton from the transient Fe+3-OOH complex. We therefore determined the crystal structure of the Fe+2-O2 complex formed by a yeast cytochrome c peroxidase mutant with Trp 191 replaced by Phe. The refined structure shows that dioxygen can form a hydrogen bond with the conserved distal His residue, but not with the conserved distal Arg residue. When the transient Fe+3-OOH complex is modelled in a similar orientation, the active site of peroxidase appears to be optimized for catalysing proton transfer between the vicinal oxygen atoms of the peroxy-anion.

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    Interaction domain for the reaction of cytochrome c with the radical and the oxyferryl heme in cytochrome c peroxidase compound I

    Miller, M.A., R.-Q. Liu, S. Hahm, L. Geren, S. Hibdon, J. Kraut, B. Durham, & F. Millett

    Biochemistry, Vol. 33, pp. 8686-8693, (1994).

    Site-directed mutants of cytochrome c peroxidase (CcP) were created to modify the interaction domain between CcP and yeast iso-1-cytochrome c (yCC) seen in the crystal structure of the CcP-yCC complex [Pelletier & Kraut (1992) Science 258, 1748-1755]. In the crystalline CcP-yCC complex, two acidic regions of CcP contact lysine residues on yCC. Mutants E32Q, D34N, E35Q, E290N, and E291Q were used to examine the effect of converting individual carboxylate side chains in the acidic regions to amides. The A193F mutant was used to test the effect of introducing a phenyl moiety at the point of closest contact between CcP and yCC in the crystal structure. Stopped-flow experiments carried out in 310 mM ionic strength buffer at pH 7 revealed that yCC initially reduced the indole radical on Trp-191 of the parent CcP compound I with a bimolecular rate constant ka = 2.5 x 10(8) M-1 s-1. A second molecule of yCC subsequently reduced the oxyferryl heme of compound II with a rate constant kb = 5 x 10(7) M-1 s-1. The bimolecular rate constants ka and kb were affected in parallel by each mutation examined. CcP mutants D34N and E290N that are closest to a complementary yCC lysine residue in the crystalline CcP-yCC complex gave the lowest values for ka and kb, which were 25-50% of the values of the CcP parent. Mutants E32Q and E291Q that are removed from the interaction domain gave the same ka and kb values as the CcP parent.

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    A cation binding motif stabilizes the compound I radical of cytochrome c peroxidase

    Miller, M.A., G.W. Han, & J. Kraut

    Proc. Natl. Acad. Sci. USA, Vol. 91, pp. 11118-11122 (1994).

    Cytochrome c peroxidase reacts with peroxide to form compound I, which contains an oxyferryl heme and an indolyl radical at Trp-191. The indolyl free radical has a half-life of several hours at room temperature, and this remarkable stability is essential for the catalytic function of cytochrome c peroxidase. To probe the protein environment that stabilizes the compound I radical, we used site-directed mutagenesis to replace Trp-191 with Gly or Gln. Crystal structures of these mutants revealed a monovalent cation binding site in the cavity formerly occupied by the side chain of Trp-191. Comparison of this site with those found in other known cation binding enzymes shows that the Trp-191 side chain resides in a consensus K+ binding site. Electrostatic potential calculations indicate that the cation binding site is created by partial negative charges at the backbone carbonyl oxygen atoms of residues 175 and 177, the carboxyl end of a long alpha-helix (residues 165-175), the heme propionates, and the carboxylate side chain of Asp-235. These features create a negative potential that envelops the side chain of Trp-191; the calculated free energy change for cation binding in this site is -27 kcal/mol (1 cal = 4.184J). This is more than sufficient to account for the stability of the Trp-191 radical, which our estimates suggest is stabilized by 7.8 kcal/mol relative to a Trp radical in solution.


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    Regulation of interprotein electron transfer by Trp 191 of cytochrome c peroxidase

    Miller, M. A., L. B. Vitello, & J. E. Erman

    Biochemistry, Vol. 34, pp. 12048-12058 (1995).

    Cytochrome c peroxidase (CcP) reacts with peroxide to form compound I, an intermediate that has an oxy-ferryl iron center and a stable indolyl radical at Trp 191. During the normal catalytic cycle, the oxy-ferryl heme and the Trp 191 radical are reduced by sequential electron transfers from ferrous cytochrome c (Cc). To investigate the role of protein structure in these electron transfer reactions, mutagenesis was used to replace Trp 191 with Phe. The Trp 191-->Phe enzyme [CcP(MI,F191)] reacts with peroxide to form an oxy-ferryl iron center and a transient porphyrin radical. The reaction of Cc from horse and yeast with peroxide-oxidized CcP(MI,F191) was characterized under transient and steady-state conditions. The rate of ET from Cc to the oxy-ferryl heme of CcP(MI,F191) was decreased by at least 10,000-fold relative to the CcP(MI) parent. This effect was observed at 20 and 100 mM ionic strength, with both yeast and horse cytochrome c as the substrate. Thus, Trp 191 is a critical component of all pathways that permit rapid reduction of the oxy-ferryl heme by Cc under these conditions. The reaction of the porphyrin radical with Cc was difficult to characterize, owing to the short half-life of this intermediate. The oxidation of Cc by this intermediate had a maximum rate constant of 32 s-1 at pH 6.0, 25 degrees C. Circumstantial evidence suggests that the porphyrin radical is not directly reduced by Cc, but is instead reduced via a protein-based radical intermediate. The steady-state activity of the mutant enzyme was 300-600-fold lower than the CcP(MI) parent, but kcat is 7-20 times greater than the rate constant for reduction of the oxy-ferryl heme under all conditions examined. Thus, the oxy-ferryl heme is not reduced to the ferric state under steady-state conditions. Transient changes in the absorption spectrum further indicate that steady-state oxidation of Cc2+ by CcP(MI,F191) occurs via reaction of peroxide with the oxy-ferryl enzyme.

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    Identifying the physiological electron transfer site of cytochrome c peroxidase by structure-based engineering

    Miller, M. A., L. Geren, G.W. Han, A. Saunders, J. Beasley, G. Pielak, B.Durham, F. Millett, & J. Kraut

    Biochemistry, Vol. 35, 667-673 (1996).

    A technique was developed to evaluate whether electron transfer (ET) complexes formed in solution by the cloned cytochrome c peroxidase [CcP(MI)] and cytochromes c from yeast (yCc) and horse (hCc) are structurally similar to those seen in the respective crystal structures. Site-directed mutagenesis was used to convert the sole Cys of the parent enzyme (Cys 128) to Ala, and a Cys residue was introduced at position 193 of CcP(MI), the point of closest contact between CcP(MI) and yCc in the crystal structure. Cys 193 was then modified with a bulky sulfhydryl reagent, 3-(N-maleimidylpropionyl)-biocytin (MPB), to prevent yCc from binding at the site seen in the crystal. The MPB modification has no effect on overall enzyme structure but causes 20-100-fold decreases in transient and steady-state ET reaction rates with yCc. The MPB modification causes only 2-3-fold decreases in ET reaction rates with hCc, however. This differential effect is predicted by modeling studies based on the crystal structures and indicates that solution phase ET complexes closely resemble the crystalline complexes. The low rate of catalysis of the MPB-enzyme was constant for yCc in buffers of 20-160 mM ionic strength. This indicates that the low affinity complex formed between CcP(MI) and yCc at low ionic strength is not reactive in ET.

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    Crystal Structure of Rat DNA Polymerase Beta: Evidence for a Common Polymerase Mechanism

    Sawaya, M., H. Pelletier, A. Kumar, S. H. Wilson, & J. Kraut

    Science Vol. 264, 1930-1935.

    Structures of the 31-kilodalton catalytic domain of rat DNA polymerase beta (polB) and the whole 39-kilodalton enzyme complexed with dATP were determined at 2.3 and 3.6 angstrom resolution respectively. The 31-kilodalton domain is composed of fingers, palm, and thumb subdomains arranged to form a DNA binding channel reminiscent of the polymerase domains of the Klenow fragment of E. coli DNA polymerase I, HIV-1 reverse transcriptase and bacteriophage T7 RNA polymerase. The NH2-terminal 8-kilodalton domain is attached to the fingers subdomain by a flexible hinge. The two invariant aspartates found in all polymerase sequences and implicated in catalytic activity have the same geometric arrangement within structurally similar but topologically distinct palms, indicating that the polymerases have maintained, or possibly reevolved, a common nucleotidyl transfer mechanism. The location of Mn+2 and dATP in polB confirms the role of the invariant aspartates in metal ion and dNTP binding.

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    Structures of Ternary Complexes of Rat DNA Polymerase Beta, a DNA Template-Primer, and ddCTP

    Pelletier, H., M. R., A. Kumar, S. H. Wilson, & J. Kraut.

    Science Vol. 264, 1891-1903.

    Two ternary complexes of rat DNA polymerase beta (polB), a DNA template-primer, and dideoxycytidine triphosphate (ddCTP) have been determined at 2.9 A and 3.6 A resolution, respectively. ddCTP is the triphosphate of dideoxycytidine (ddC), a nucleoside analog that targets the reverse transcriptase of human immunodeficiency virus (HIV) and is at present used to treat AIDS. Although crystals of the two complexes belong to different space groups, the structures are similar, suggesting that the polymerase-DNA-ddCTP interactions are not affected by crystal packing forces. In the polB active site, the attacking 3'-OH of the elongating primer, the ddCTP phosphates, and two Mg+2 ions are all clustered around Asp190, Asp192, and Asp256. Two of these residues, Asp190 and Asp256, are present in the amino acid sequences of all polymerases so far studied and are also spatially similar in the four polymerases--the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, T7 RNA polymerase, and rat DNA polB--whose crystal structures are now known. A two-metal ion mechanism is described for the nucleotidyltransfer reaction and may apply to all polymerases. In the ternary complex structures analyzed, polB binds to the DNA template-primer in a different manner from that recently proposed for other polymerase-DNA models.

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    Isomorphous crystal structures of Escherichia coli dihydrofolate reductase complexed with folate, deazafolate, and 5,10-dideazatetrahydrofolate: mechanistic implications

    Reyes, V. M., M. R. Sawaya, K. A> Brown, & J. Kraut

    Biochemistry, Vol. 34, pp. 2710-2723, (1995).

    Crystal structures of Escherichia coli dihydrofolate reductase (ecDHFR, EC 1.5.1.3) in binary complexes with folate, 5-deazafolate (5dfol), and 5,10-dideazatetrahydrofolate (ddTHF) have been refined to R-factors of 13.7%, 14.9%, and 14.5%, respectively, all at 1.9 A. All three are isomorphous with a previously reported binary complex of ecDHFR with methotrexate (MTX), in space group P6(1), two molecules per asymmetric unit [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662]. A hitherto unobserved water molecule is hydrogen bonded to the pteridine N5 and O4 in both molecules of the asymmetric unit of the folate complex (but not the 5dfol or ddTHF complexes), supporting the hypothesis that N5 protonation of bound substrate, an important step of the DHFR reaction, occurs by way of such a water molecule. There is no indication of a hydrogen bond between N8 of 5dfol and the backbone carbonyl of Ile-5, suggesting that the bacterial enzyme, unlike the human enzyme [Davies, J. F., II, Delcamp, T. J., Prendergast, N. J., Ashford, V. A., Freisheim, J. H., & Kraut, J. (1990) Biochemistry 29, 9467-9479], does not favor protonation at N8. Perhaps this explains why bacterial DHFR is much less effective than vertebrate DHFR in folate reduction. When the ecDHFR.NADPH complex (space group P3221; M. R. Sawaya, in preparation) is superimposed on the folate and 5dfol complexes, the distances from pteridine C6 to nicotinamide C4 were found to be 2.9 and 2.8 A, respectively, in close agreement with the theoretically calculated optimal distance in the transition state for hydride transfer [Wu, Y. D., & Houk, K. N. (1987) J. Am. Chem. Soc. 109, 906-908, 2226-2227]. In contrast to the planar ring system of folate or 5dfol, the reduced pteridine ring of ddTHF is severely puckered and bent toward the nicotinamide pocket, with the reduced pyridine ring assuming a half-chair type of conformation. This change in shape causes the pteridine ring to bind with O4 closer to Trp-22(N epsilon 1) by over 0.5 A, so that an invariant water molecule now bridges these two atoms with ideal hydrogen bonds. Furthermore, while the pABA rings of folate and 5dfol are nearly coincident and closer to the alpha C helix than to the alpha B helix, those of MTX and ddTHF are displaced along the binding crevice by approximately 1.1 and 0.6 A, respectively, and are equidistant from alpha B and alpha C. Under construction.

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    Crystal structures of Escherichia coli dihydrofolate reductase complexed with 5-formyltetrahydrofolate (folinic acid) in two space groups - evidence for enolization of pteridine O4

    Lee, H., V. M. Reyes, & J. Kraut

    Biochemistry Vol. 35, pp. 7012-7020, (1996).

    Under construction.

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