Solution 1H NMR of the molecular and electronic structure of the heme cavity and substrate binding pocket of high-spin ferric horseradish peroxidase: effect of His42Ala mutation.
Solution 1H NMR has been used to assign a major portion of the heme environment and the substrate-binding pocket of resting state horseradish peroxidase, HRP, despite the high-spin iron(III) paramagnetism, and a quantitative interpretive basis of the hyperfine shifts is established. The effective assignment protocol included 2D NMR over a wide range of temperatures to locate residues shifted by paramagnetism, relaxation analysis, and use of dipolar shifts predicted from the crystal structure by an axial paramagnetic susceptibility tensor normal to the heme. The most effective use of the dipolar shifts, however, is in the form of their temperature gradients, rather than by their direct estimation as the difference of observed and diamagnetic shifts. The extensive assignments allowed the quantitative determination of the axial magnetic anisotropy, Deltachi(ax) = -2.50 x 10(-8) m(3)/mol, oriented essentially normal to the heme. The value of Deltachi(ax) together with the confirmed T(-2) dependence allow an estimate of the zero-field splitting constant D = 15.3 cm(-1), which is consistent with pentacoordination of HRP. The solution structure was generally indistinguishable from that in the crystal (Gajhede, M.; Schuller, D. J.; Henriksen, A.; Smith, A. T.; Poulos, T. L. Nature Structural Biology 1997, 4, 1032-1038) except for Phe68 of the substrate-binding pocket, which was found turned into the pocket as found in the crystal only upon substrate binding (Henriksen, A.; Schuller, D. J.; Meno, K.; Welinder, K. G.; Smith, A. T.; Gajhede, M. Biochemistry 1998, 37, 8054-8060). The reorientation of several rings in the aromatic cluster adjacent to the proximal His170 is found to be slow on the NMR time scale, confirming a dense, closely packed, and dynamically stable proximal side up to 55 degrees C. Similar assignments on the H42A-HRP mutant reveal conserved orientations for the majority of residues, and only a very small decrease in Deltachi(ax) or D, which dictates that five-coordination is retained in the mutant. The two residues adjacent to residue 42, Ile53 and Leu138, reorient slightly in the mutant H42A protein. It is concluded that effective and very informative 1H NMR studies of the effect of either substrate binding or mutation can be carried out on resting state heme peroxidases.
Asokan
A
A
Department of Chemistry and NMR Facility, University of California, One Shields Avenue, Davis, California 95616, USA.
de Ropp
J S
JS
Newmyer
S L
SL
Ortiz de Montellano
P R
PR
La Mar
G N
GN
D000465:Algorithms; D019943:Amino Acid Substitution; D016880:Anisotropy; D006418:Heme / Q000737:chemistry* / Q000235:genetics; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D012996:Solutions
D012996:Solutions; D006418:Heme; D006735:Horseradish Peroxidase