Solution H-1 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 H-1 NMR has been used to assign a major portion of the heme enviro nment and the substrate-binding pocket of resting state horseradish peroxid ase, HRP, despite the high-spin iron(m) paramagnetism, and a quantitative i nterpretive basis of the hyperfine shifts is established. The effective ass ignment protocol included 2D NMR over a wide range of temperatures to locat e residues shifted by paramagnetism, relaxation analysis, and use of dipola r shifts predicted from the crystal structure by an axial paramagnetic susc eptibility tenser normal to the heme. The most effective use of the dipolar shifts, however, is in the form of their temperature gradients, rather tha n by their direct estimation as the difference of observed and diamagnetic shifts. The extensive assignments allowed the quantitative determination of the axial magnetic anisotropy Delta chi (ax) = -2.50 x 10(-8) m(3)/mol, or iented essentially normal to the heme. The value of Delta chi (ax) together with the confirmed T-2 dependence allow an estimate of the zero-field spli tting 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.; Po ulos, 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, hi. Biochemistry 1 998, 37, 8054-8060). The reorientation of several rings in the aromatic clu ster adjacent to the proximal His170 is found to be slow on the NMR time sc ale, confirming a dense, closely packed, and dynamically stable proximal si de up to 55 degreesC. Similar assignments on the H42A-HRP mutant reveal con served orientations for the majority of residues, and only a very small dec rease in Delta chi (ax) or D, which dictates that five-coordination is reta ined in the mutant. The two residues adjacent to residue 42, Ile53 and Leu1 38, reorient slightly in the mutant H42A protein. It is concluded that effe ctive and very informative H-1 NMR studies of the effect of either substrat e binding or mutation can be carried out on resting state heme peroxidases.