[NMR paper] 2D NMR of paramagnetic metalloenzymes: cyanide-inhibited horseradish peroxidase.

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2D NMR of paramagnetic metalloenzymes: cyanide-inhibited horseradish peroxidase.

Related Articles 2D NMR of paramagnetic metalloenzymes: cyanide-inhibited horseradish peroxidase.

J Biomol NMR. 1991 Jul;1(2):175-90

Authors: de Ropp JS, Yu LP, La Mar GN

Two-dimensional (2D) proton NMR correlation spectroscopy, COSY, and nuclear Overhauser spectroscopy, NOESY, have been used to explore the applicability of these methods for the moderately large (42 KDa), paramagnetic cyanide-inhibited derivative of horseradish peroxidase, HRP-CN. The target resonances are those in the active site of HRP-CN which experience substantial hyperfine shifts and paramagnetic relaxation. The magnitude COSY experiment was found to yield cross peaks for all known spin-coupled heme substituents, as well as for the majority of non-heme hyperfine shifted protons, in spite of line widths of the order of approximately 100 Hz. Moreover, the rapid relaxation of the hyperfine-shifted resonances allows the extremely rapid collection of useful 2D NMR data sets without the loss of information. For the heme, the combination of COSY cross peaks for the vinyl and propionate substituents, and NOESY cross peaks among these substituent protons and heme methyls, allows assignment of heme resonances without recourse to deuterium labeling of the heme. A seven-proton coupled spin system was identified in the upfield region that is consistent with originating from the proposed catalytic Arg38 residue in the distal heme pocket, with orientation relative to the heme similar to that found in cytochrome c peroxidase. The upfield hyperfine-shifted methyl group in the substrate binding pocket previously proposed to arise from Leu237 is shown to arise instead from an as yet unidentified Ile. NOESY spectra collected at very short (3 ms) and intermediate (20 ms) mixing times indicate that build-up curves can be obtained that should yield estimates of distances in the heme cavity. It is concluded that 2D NMR studies should be able to provide the heme assignments, aid in identifying the catalytic residues, and provide information on the spatial disposition of such residues in the active site for cyanide complexes of a number of intermediate to large paramagnetic heme peroxidases, as well as for other paramagnetic metalloenzymes with line widths of approximately 100 Hz. Moreover, paramagnetic-induced hyperfine shifts and linewidths to approximately 100 Hz need not interfere with the complete solution structure determination of a small paramagnetic protein solely on the basis of 2D NMR data.

PMID: 1841693 [PubMed - indexed for MEDLINE]



Source: PubMed