Proton NMR assignments of heme contacts and catalytically implicated amino acids in cyanide-ligated cytochrome c peroxidase determined from one-and two-dimensional nuclear Overhauser effects

Abstract

Proton NMR assignments of the heme pocket and catalytically relevant amino acid protons have been accomplished for cyanide-ligated yeast cytochrome c peroxidase. This form of the protein, while not enzymatically active itself, is the best model available (that displays a resolvable proton NMR spectrum) for the six-coordinate low-spin active intermediates, compounds I and II. The assignments were made with a combination of one- and two-dimensional nuclear Overhauser effect methods and demonstrate the utility of NOESY experiments for paramagnetic proteins of relatively large size (Mr 34,000). Assignments of both isotope exchangeable and nonexchangeable proton resonances were obtained by using enzyme preparations in both 90% H2O/10% D2O and, separately, in 99.9% D2O solvent systems. Complete resonance assignments have been achieved for the proximal histidine, His-175, and His-52, which is a member of the catalytic triad on the distal side of the heme. In addition, partial assignments are reported for Trp-51 and Arg-48, catalytically important residues, both on the distal side. Aside from His-175, partial assignments for amino acids on the proximal side of the heme are proposed for the alanines at primary sequence positions 174 and 176 and for Thr-180 and Leu-232.

The Effect of the Asn82→Asp Mutation in Yeast Cytochrome c Peroxidase Studied by Proton NMR Spectroscopy

“…There is more uncertainty in assigning the heme 8p protons for [N82D]cytochrome c peroxidase-CN due to the crowded spectrum downfield from -4 ppm, but if these ten- tative 8-vinyl assignments (Table 1) are accurate then the NOE connectivity pattern indicates that the orientation of the heme 8-vinyl group is similar to that shown in Fig. 1C and also similar to that demonstrated for the wild-type cyanideligated enzyme [22]. What is not obvious in our NOESY spectra is the set of connectivities for Arg48 protons (Fig.…”

“…What is not obvious in our NOESY spectra is the set of connectivities for Arg48 protons (Fig. 1) that was found in this low-frequency region for the wild-type cyanide-ligated enzyme [22]. Changes in chemical shifts and the broadness of the [N82D]cytochrome c peroxidase-CN resonances make it impossible to identify good candidates for the Arg48 spin system.…”

“…Apparent non-selective spin-lattice relaxation times T,app for [N82D]cytochrome c peroxidase-CN are derived from linear least squares analysis of initial slopes of semi-log plots of inversion-recovery data (correlation coefficients better than 0.99) The first number in parentheses is the uncertainty from two separate inversion-recovery experiments, whereas the second number in parentheses is the time (ms) of initial slope over which the calculation was carried out. The assignments and shifts for wild-type cytochrome c peroxidase-CN are from [22] and the labels correspond to Fig. 2C Standard NMR sample conditions [15,221 included solution pH of pH 6.00-pH 7.20, and calibrated (with a methanol sample) probe temperatures of 5 3 5°C .…”

“…On the basis of the spectral similarities shown in Fig. 2, initial proton assignments for [N82D]cytochrome c peroxidase-CN could by postulated based on the evident correspondence with the wild-type cyanide-ligated enzyme [22,261. The NOESY connectivities for [N82D]cytochrome c peroxidase-CN confirm most of this correspondence (Fig.…”

“…5E. The exact identity of resonance E is uncertain, but it is apparently derived from either Trp5l [22] or Arg48 [26] (Fig. 1).…”