title: Expression, purification, characterization, and NMR studies of highly deuterated recombinant cytochrome c peroxidase.
abstract: Two forms of extensively deuterated S. cerevisiae cytochrome c peroxidase (CcP; EC 1.11.1.5) have been overexpressed in E. coli by growth in highly deuterated medium. One of these ferriheme enzyme forms (recDCcP) was produced using >97% deuterated growth medium and was determined to be approximately 84% deuterated. The second form [recD(His)CcP] was grown in the same highly deuterated medium that had been supplemented with excess histidine (at natural hydrogen isotope abundance) and was also approximately 84% deuterated. This resulted in direct histidine incorporation without isotope scrambling. Both of these enzymes along with the corresponding recombinant native CcP (recNATCcP), which was expressed in a standard medium with normal hydrogen isotope abundance, consisted of 294 amino acid polypeptide chains having the identical sequence to the yeast-isolated enzyme, without any N-terminal modifications. Comparative characterizations of all three enzymes have been carried out for the resting-state, high-spin forms and in the cyanide-ligated, low-spin forms. The primary physical methods employed were electrophoresis, UV-visible spectroscopy, hydrogen peroxide reaction kinetics, mass spectrometry, and (1)H NMR spectroscopy. The results indicate that high-level deuteration does not significantly alter CcP's reactivity or spectroscopy. As an example of potential NMR uses, recDCcPCN and recD(His)CcPCN have been used to achieve complete, unambiguous, stereospecific (1)H resonance assignments for the heme hyperfine-shifted protons, which also allows the heme side chain conformations to be assessed. Assigning these important active-site protons has been an elusive goal since the first NMR spectra on this enzyme were reported 18 years ago, due to a combination of the enzyme's comparatively large size, paramagnetism, and limited thermal stability.
authors.affiliation: Department of Chemistry, Laboratory for Biotechnology and BioAnalysis, and NMR Spectroscopy Center, Washington State University, Pullman, Washington 99164-4630, USA.
authors.forename: M I
authors.initials: MI
authors.lastname: Savenkova
authors.forename: J D
authors.initials: JD
authors.lastname: Satterlee
authors.forename: J E
authors.initials: JE
authors.lastname: Erman
authors.forename: W F
authors.initials: WF
authors.lastname: Siems
authors.forename: G L
authors.initials: GL
authors.lastname: Helms
mesh_terms: D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006418:Heme / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011522:Protons; D011994:Recombinant Proteins; D006418:Heme; D003578:Cytochrome-c Peroxidase