The reaction of ferric cytochrome c peroxidase (CcP) from Saccharomyces cerevisiae with peroxide produces compound I, characterized by both an oxyferryl iron center and a protein-based free radical. The electron paramagnetic resonance (EPR) signal of the CcP compound I radical can be resolved into a broad majority component which accounts for approximately 90% of the spin intensity and a narrow minority component which accounts for approximately 10% of the integrated spin intensity [Hori, H., & Yonetani, T. (1985) J. Biol. Chem. 260, 3549-3555]. It was shown previously that the broad component of the compound I radical signal is eliminated by mutation of Trp-191 to Phe [Scholes, C. P., Liu, Y., Fishel, L. F., Farnum, M. F., Mauro, J. M., & Kraut, J. (1989) Isr. J. Chem. 29, 85-92]. The present work probed the effect of mutations in the vicinity of this residue by EPR and electron-nuclear double resonance (ENDOR). These mutations were obtained from a plasmid-encoded form of S. cerevisiae expressed in Escherichia coli [Fishel, L. A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360]. The EPR line shape and ENDOR signals of the compound I radical were perturbed only by mutations that alter Trp-191 or residues in its immediate vicinity: namely, Met-230 and Met-231, which have sulfur atoms within 4 A of the indole ring, and Asp-235, which forms a hydrogen bond with the indole nitrogen of Trp-191. Mutations of other potential oxidizable sites (tryptophan, tyrosine, methionine, and cysteine) did not alter the EPR line shapes of the compound I radical, although the integrated spin intensities were weaker in some of these mutants. Mutations at Met-230 and/or -231 perturbed the EPR line shapes of the compound I radical signal but did not eliminate it. ENDOR of these two methionine mutants showed alteration to the hyperfine couplings of several strongly coupled protons, which are characteristic of the majority compound I radical electronic structure, and a change in weaker hyperfine couplings, which suggests a different orientation of the radical with respect to its surroundings in the presence of these methionine mutations. Besides the Trp-191----Phe mutation, only the Asp-235----Asn mutation eliminated the broad component of the compound I signal. Loss of the broad compound I EPR signal coincides with both the loss of the Asp----Trp-191 hydrogen-bonding interaction and alteration of the position of the indole ring of Trp-191.(ABSTRACT TRUNCATED AT 400 WORDS)

The pH dependence of steady-state parameters for [1,1-1H2]- and [1,1-2H2]benzylamine oxidation and of tritium exchange from [2-3H]dopamine has been measured in the bovine plasma amine oxidase reaction. Deuterium isotope effects on kcat/Km for benzylamine are observed to be constant, near the intrinsic value of 13.5, over the experimental pH range, indicating that C-H bond cleavage is fully rate limiting for this parameter. As a consequence, pKa values derived from kcat/Km profiles, 8.0 +/- 0.1 (pK1) and 9.0 +/- 0.16 (pKs), can be ascribed to microscopic pKa values for the ionization of an essential active site residue (EB1) and substrate, respectively. Profiles for kcat and Dkcat show that EB1 undergoes a perturbation from 8.0 to 5.6 +/- 0.3 (pK1') in the presence of substrate; additionally, a second ionization, pK2 = 7.25 +/- 0.25, is observed to mediate but not be essential for enzyme reoxidation. The pH dependence of the ratio of tritium exchange to product formation for dopamine also indicates base catalysis with a pKexch = 5.5 +/- 0.01, which is within experimental error of pK1'. We conclude that the data presented herein support a single residue catalyzing both substrate oxidation and exchange, consistent with recent stereochemical results that implicate a syn relationship between these processes [Farnum, M., & Klinman, J.P. (1985) Fed. Proc., Fed. Am. Soc. Exp. Biol. 44, 1055]. This conclusion contrasts with earlier kinetic data in support of a large rate differential for the exchange of hydrogen from C-1 vs. C-2 of phenethylamine derivatives [Palcic, M.M., & Klinman, J.P. (1983) Biochemistry 22, 5957-5966].(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract. EPR, ENDOR and pulsed EPR were applied to the radical signals of yeast cytochrome c peroxidase-compound I from this protein expressed in E. coli and two variants prepared after site-directed mutagenesis. The compound I complexes studied were first, that of the parent peroxidase expressed in E. coli, and next, those of the following amino acid mutants as specific probes for the radical in compoundI: 1. Tryptophan-51~Phenylalanine; 2. Tryptophan-l91~Phenylalan-ine. Tryptophan-51 lies near the heme on the distal (H,.Oz-binding) side of cytochrome c peroxidase, while Tryptophan-191 lies in contact with the proximal heme ligand, Histidine-175. EPR above 20 K and saturation recovery T J measurements at liquid He temperatures showed the unusual temperature-dependent relaxation of compound I's axially symmetric signal (gil =2.035, gJ. =2.00), which has comprised the major radical species in bakers' yeast wildtype compound I and here in all but the Tryptophan-191~Phenylalanine mutant. The ENDOR of the major radical species was characterized by the substantial hyperfine couplings previously proposed to be at a sulfur-based radical. The mutation Tryptophan-51~Phenyla-lanine does not perturb these ENDOR features. Pulse field-sweep EPR resolved for the first time distinct proton hyperfme couplings due to the features observed by ENDOR. A narrow, more typical organic free radical signal at g = 2.001 with resolved hyperfine structure was the only species observed from the Phenylalanine mutant and was a minor feature in all samples. The Tryptophan191~Phenylalanine mutation does perturb or destabilize the majority radical species of compound I. Thus, Tryptophan-l91 is either intimately involved with the kinetics of radical formation or participates intimately in the majority radical site, despite not being a sulfur-containing amino acid.

The adaptability of Escherichia coli dihydrofolate reductase (DHFR) is being explored by identifying second-site mutations that can partially suppress the deleterious effect associated with removal of the active-site proton donor aspartic acid-27. The Asp27----serine mutant DHFR (D27S) was previously characterized and the catalytic activity found to be greatly decreased at pH 7.0 [Howell et al. (1986) Science 231, 1123-1128]. Using resistance to trimethoprim (a DHFR inhibitor) in a genetic selection procedure, we have isolated a double-mutant DHFR gene containing Asp27----Ser and Phe137----Ser mutations (D27S+F137S). The presence of the F137S mutation increases kcat approximately 3-fold and decreases Km(DHF) approximately 2-fold over D27S DHFR values. The overall effect on kcat/Km(DHF) is a 7-fold increase. The D27S+F137S double-mutant DHFR is still 500-fold less active than wild-type DHFR at pH 7. Surprisingly, Phe137 is approximately 15 A from residue 27 in the active site and is part of a beta-bulge. We propose the F137S mutation likely causes its catalytic effect by slightly altering the conformation of D27S DHFR. This supposition is supported by the observation that the F137S mutation does not have the same kinetic effect when introduced into the wild-type and D27S DHFRs, by the altered distribution of two conformers of free enzyme [see Dunn et al. (1990)] and by a preliminary difference Fourier map comparing the D27S and D27S+F137S DHFR crystal structures.

A remarkable correlation has been discovered between fluorescence lifetimes of bound NADPH and rates of hydride transfer among mutants of dihydrofolate reductase (DHFR) from Escherichia coli. Rates of hydride transfer from NADPH to dihydrofolate change by a factor of 1,000 for the series of mutant enzymes. Since binding constants for the initial complex between coenzyme and DHFR change by only a factor of 10, the major portion of the change in hydride transfer must be attributed to losses in transition-state stabilization. The time course of fluorescence decay for NADPH bound to DHFR is biphasic. Lifetimes ranging from 0.3 to 0.5 ns are attributed to a solvent-exposed dihydronicotinamide conformation of bound coenzyme which is presumably not active in catalysis, while decay times (tau 2) in the range of 1.3 to 2.3 ns are assigned to a more tightly bound species of NADPH in which dihydronicotinamide is sequestered from solvent. It is this slower component that is of interest. Ternary complexes with three different inhibitors, methotrexate, 5-deazafolate, and trimethoprim, were investigated, along with the holoenzyme complex; 3-acetylNADPH was also investigated. Fluorescence polarization decay, excitation polarization spectra, the temperature variation of fluorescence lifetimes, fluorescence amplitudes, and wavelength of absorbance maxima were measured. We suggest that dynamic quenching or internal conversion promotes decay of the excited state in NADPH-DHFR. When rates of hydride transfer are plotted against the fluorescence lifetime (tau 2) of tightly bound NADPH, an unusual correlation is observed. The fluorescence lifetime becomes longer as the rate of catalysis decreases for most mutants studied. However, the fluorescence lifetime is unchanged for those mutations that principally alter the binding of dihydrofolate while leaving most dihydronicotinamide interactions relatively undisturbed. The data are interpreted in terms of possible dynamic motions of a flexible loop region in DHFR which closes over both substrate and coenzyme binding sites. These motions could lead to faster rates of fluorescence decay in holoenzyme complexes and, when correlated over time, may be involved in other motions which give rise to enhanced rates of catalysis in DHFR.