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Electron spin density on the axial His ligand of high-spin and low-spin nitrophorin 2 probed by heteronuclear NMR spectroscopy.

ABSTRACT: The electronic structure of heme proteins is exquisitely tuned by the interaction of the iron center with the axial ligands. NMR studies of paramagnetic heme systems have been focused on the heme signals, but signals from the axial ligands have been rather difficult to detect and assign. We report an extensive assignment of the (1)H, (13)C and (15)N resonances of the axial His ligand in the NO-carrying protein nitrophorin 2 (NP2) in the paramagnetic high-spin and low-spin forms, as well as in the diamagnetic NO complex. We find that the high-spin protein has ? spin delocalization to all atoms in the axial His57, which decreases in size as the number of bonds between Fe(III) and the atom in question increases, except that within the His57 imidazole ring the contact shifts are a balance between positive ? and negative ? contributions. In contrast, the low-spin protein has ? spin delocalization to all atoms of the imidazole ring. Our strategy, adequately combined with a selective residue labeling scheme, represents a straightforward characterization of the electron spin density in heme axial ligands.

SUBMITTER: Abriata LA

PROVIDER: S-EPMC3594510 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Electron spin density on the axial His ligand of high-spin and low-spin nitrophorin 2 probed by heteronuclear NMR spectroscopy.

Abriata Luciano A LA Zaballa María-Eugenia ME Berry Robert E RE Yang Fei F Zhang Hongjun H Walker F Ann FA Vila Alejandro J AJ

Inorganic chemistry 20130117 3

The electronic structure of heme proteins is exquisitely tuned by the interaction of the iron center with the axial ligands. NMR studies of paramagnetic heme systems have been focused on the heme signals, but signals from the axial ligands have been rather difficult to detect and assign. We report an extensive assignment of the (1)H, (13)C and (15)N resonances of the axial His ligand in the NO-carrying protein nitrophorin 2 (NP2) in the paramagnetic high-spin and low-spin forms, as well as in th ...[more]

PMID: 23327568

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Project description:Nitrophorin 3 (NP3) is the only one of the four major NO-binding heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus (also called the Kissing Bug) for which it has not been possible to obtain crystals of diffraction quality for structure determination by X-ray crystallography. Thus we have used NMR spectroscopy, mainly of the hyperfine-shifted ferriheme substituent resonances, to learn about the similarities and differences in the heme pocket and the iron active site of NP3 as compared to NP2, which has previously been well-characterized by both X-ray crystallography and NMR spectroscopy. Only one residue in the heme pocket differs between the two, F27 of NP2 is Y27 for NP3; in both cases this residue is expected to interact strongly with the 2-vinyl side chain of the B heme rotational isomer or the 4-vinyl of the A heme rotational isomer. Both the high-spin (S = 5/2) aquo complex, NP3-H(2)O, and the low-spin (S = 1/2) N-methylimidazole (NMeIm) complex of NP3 have been studied. It is found that the chemical shifts of the protons of both forms are similar to those of the corresponding NP2 complexes, but with minor differences that indicate a slightly different angle for the proximal histidine (H57) ligand plane. The B heme rotational isomer is preferred by both NP3 and NP2 in both spin states, but to a greater extent when phenylalanine is present at position 27 (A:B = 1:8 for NP2, 1:6 for NP3-Y27F, 1:4 for NP3, and 1:3 for NP2-F27Y). Careful analysis of the 5Me and 8Me shifts of the A and B isomers of the two high-spin nitrophorins leads to the conclusion that the heme environment for the two isomers differs in some way that cannot be explained at the present time. The kinetics of deprotonation of the high-spin complexes of NP2 and NP3 are very different, with NP2 giving well-resolved high-spin aquo and "low-spin" hydroxo proton NMR spectra until close to the end of the titration, while NP3 exhibits broadened (1)H NMR spectra indicative of an intermediate rate of exchange on the NMR timescale between the two forms throughout the titration. The heme methyl shifts of NP2-OH are similar in magnitude and spread to those of NP2-CN, while those of metmyoglobin-hydroxo complexes are much larger in magnitude but not spread. It is concluded that the hydroxo complex of NP2 is likely S = 1/2 with a mixed (d(XY))(2)(d(XZ),d(YZ))(3)/(d(xy))(1)(d(xz),d(yz))(4) electron configuration, while those of met-Mb-OH are likely S = 1/2,3/2 mixed spin systems.

Dataset Information

Electron spin density on the axial His ligand of high-spin and low-spin nitrophorin 2 probed by heteronuclear NMR spectroscopy.

Publications

Electron spin density on the axial His ligand of high-spin and low-spin nitrophorin 2 probed by heteronuclear NMR spectroscopy.

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