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)

Articles you may be interested inA multifrequency high-field pulsed electron paramagnetic resonance/electron-nuclear double resonance spectrometer Rev. Sci. Instrum. 79, 064703 (2008); 10.1063/1.2937630 17O hyperfine and quadrupole interactions for water ligands in frozen solutions of high spin Mn2+

Electron nuclear double resonance (ENDOR) and the related pulse technique of pulse field sweep EPR (PFSEPR) were used to probe the site I environment of Mn2+ in the oxalate-ATP complex of pyruvate kinase. Assignment of features and an estimate of hyperfine couplings have shown proximity of protons to the metal ions through their dipolar interaction and proximity of 31P and 17O because of a contact interaction from direct Mn(2+)-ligand covalent spin transfer. Since Mn2+ is a spin5/2 ion whose Ms = +/- 1/2, +/- 3/2, and +/- 5/2 electron spin states can all contribute to EPR and ENDOR, we have developed experimental and theoretical strategies for elucidating hyperfine couplings to the Mn2+ electron spin states. Solvent-exchangeable proton ENDOR features were evident with couplings very similar to the hyperfine couplings of H2O in [Mn(H2O)6]2+. ENDOR of exchangeable, more distant protons originated from a dipolar coupling such as could be expected from protons residing 5.5 A from Mn2+ and hydrogen-bonded to a nonliganding oxygen or nitrogen. Nonexchangeable proton ENDOR features indicated dipolar coupling to proton(s) from the protein residing at approximately 4.5 A from the Mn2+. The approximately 4-MHz 31P phosphate hyperfine couplings in Mn(II)-nucleotide models and in pyruvate kinase were similar, but a detailed ENDOR and PFSEPR comparison revealed that the hyperfine coupling to the ATP gamma-phosphate in pyruvate kinase was approximately 10% less than coupling to phosphates of Mn(II)-nucleotides. [In pyruvate kinase only the gamma-phosphate has been shown to bind to Mn2+ at site I (Lodato & Reed, 1987).](ABSTRACT TRUNCATED AT 250 WORDS)

Articles you may be interested inPulsed and continuous wave electron nuclear double resonance patterns of aquo protons coordinated in frozen solution to high spin MN2+ J. Chem. Phys. 98, 5147 (1993); 10.1063/1.464917 Multifrequency electron spinecho envelope modulation: The determination of nitro group 1 4N hyperfine and quadrupole interactions of DPPH in frozen solution The magnetic couplings of 17 O in H 2 17O coordinated to high spin Mn 2ϩ in a frozen aqueous solution were determined using the complementary magnetic resonance techniques of pulsed and continuous wave ͑cw͒ ENDOR ͑electron nuclear double resonance͒, ESEEM ͑electron spin echo envelope modulation͒, and PFSEPR ͓pulse field sweep electron paramagnetic resonance ͑EPR͔͒. Several complications arise from the high electron spin multiplicity of the d 5 , Mn 2ϩ ion and the high nuclear spin multiplicity, Iϭ5/2, of the 17 O nucleus. At the applied magnetic field strengths in 9 GHz EPR studies, the zero-field splitting of the Sϭ5/2 Mn 2ϩ ion in aqueous frozen solution is small relative to the electron spin Zeeman interaction so that the M S ϭϮ1/2,Ϯ3/2,Ϯ5/2 electron spin states all contribute to the ENDOR spectrum. This results in a complex spectrum in which the 17 O ENDOR powder pattern arising from the M S ϭϮ1/2 manifolds are separately resolved but the powder patterns from the M S ϭϮ3/2,Ϯ5/2 manifolds overlap the multiple 1 H ENDOR lines arising from all six M S manifolds ͓X. Tan, M. Bernardo, H. Thomann, and C. P. Scholes, J. Chem. Phys. 98, 5147 ͑1993͔͒. Given this complexity, a combination of complementary spectroscopic techniques and numerical simulations are used to deconvolute the overlapping spectra and to assign the spectral lines. The ENDOR spectra provided an experimental description of H 2 17 O hyperfine couplings to high spin Mn 2ϩ in a frozen solution. The ESEEM results are consistent with the first-order assignments of the ENDOR lines and demonstrate the feasibility of ESEEM measurements of 17 O ligand hyperfine couplings to Mn 2ϩ . Simulations of the 17 O ENDOR hyperfine patterns of aqueous frozen solutions of Mn 2ϩ , especially those near 20 MHz, indicated an A-tensor anisotropy of A Ќ ϭϪ6.5Ϯ0.5 MHz and A ʈ ϭϪ9.5Ϯ0.5 MHz, consistent with couplings observed by single crystal ENDOR of H 2 17O ligated to Mn 2ϩ doped in ͓LaMg͑NO 2 ͒ 12 •24͑H 2 O͔͒. More detailed simulations of the ENDOR pattern below 10 MHz indicated the need for quadrupole couplings consistent with those measured by single crystal ENDOR and with those determined by gas phase measurements on H 17 OD. Simulations of the ENDOR spectra recorded by the cw and pulsed techniques have delineated important features of the techniques which must be taken into account for a quantitative analysis of the ENDOR amplitudes. It is expected that the general ENDOR conditions employed and the theory developed will be useful in frozen solution studies of 17 O involved as a ligand to Mn 2ϩ in enzymes.