Characterization of the active site in nickel hydrogenase and the Trp pi-cation radical in cytochrome c peroxidase using advanced electron paramagnetic spectroscopies

The fully oxidized state of cytochrome c peroxidase (CcP), ES, contains two oxidizing equivalents, one as an oxy-ferryl heme (S = 1) and the other is proposed as an organic radical located on Trp 191 (S = 1/2). The EPR spectrum of ES has been shown to be due to a weak, distributed exchange coupling between the two paramagnetic redox centers. CW and Pulsed Q-Band ENDOR on samples enriched with {dollar}rmsp{lcub}13{rcub}C, sp{lcub}15{rcub}N,{dollar} and {dollar}sp2{dollar}H tryptophan are reported. An exchangeable proton with A({dollar}rmsp1H) approx 16{dollar} MHz is associated with N1-H of the Trp {dollar}pi{dollar}-cation radical and not with C2-H. Through a combination of {dollar}sp{lcub}13{rcub}{dollar}C and {dollar}sp2{dollar}H labeling, the hyperfine couplings at g = 2.01 for {dollar}rmsp{lcub}13{rcub}C(beta{dollar}), and the {dollar}rm C(beta)Hsb2,{dollar} N1H, and C2H protons have been determined. Analysis of these couplings has yielded spin densities for the radical in agreement with those predicted for the cation radical. This confirms previous assignments as a Trp {dollar}pi{dollar}-cation radical which is not perturbed by N1-H{dollar}cdots{dollar}O hydrogen bonding to the carboxylate of Asp 235.; Q-band Pulsed ENDOR and X-band ESEEM techniques are used to determine the hyperfine tensors for ethylene and cyano carbons, and N, of bis-(maleonitrile-dithiolato)nickel(III) ( (Ni(mnt){dollar}sb2rbracksp-).{dollar} These measurements give {dollar}pi{dollar}-electron spin densities such that in total, {dollar}sim{dollar}0.15 of the spin resides on the ligand atoms C and N, while the rest resides in the NiS{dollar}sb4{dollar} core. These results are compared with MO calculations.; X-ray crystallographic analysis showed that the active site of Desulfovibrio gigas (Dg) Nickel Hydrogenase, known to contain Ni, actually contains a hetero-dinuclear cluster consisting of nickel and a second transition-metal ion proposed to be iron. {dollar}sp{lcub}57{rcub}{dollar}Fe Q-band Mims pulsed ENDOR study of {dollar}sp{lcub}57{rcub}{dollar}Fe-enriched Dg hydrogenase in NiA and catalytically active NiC forms are reported and compared with {dollar}sp{lcub}57{rcub}{dollar}Fe enriched D. desulfuricans ATCC 27774 hydrogenase in the NiB form. In NiA, {dollar}sp{lcub}57{rcub}{dollar}Fe ENDOR spectra shows a small isotropic coupling of A({dollar}sp{lcub}57{rcub}{dollar}Fe) {dollar}sim{dollar} 1 MHz and indicate that the cluster contains an iron ion in the low-spin (S = 0) ferrous state. This hyperfine coupling is lost in the NiB and NiC states.