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1H and 13C NMR spectroscopic studies of the ferriheme resonances of three low-spin complexes of wild-type nitrophorin 2 and nitrophorin 2(V24E) as a function of pH.

ABSTRACT: The ferriheme resonances of the low-spin (S = 1/2) complexes of wild-type (wt) nitrophorin 2 (NP2) and its heme pocket mutant NP2(V24E) with imidazole (ImH), histamine (Hm), and cyanide (CN(-)) as the sixth ligand have been investigated by NMR spectroscopy as a function of pH (4.0-7.5). For the three wt NP2 complexes, the ratio of the two possible heme orientational isomers, A and B, remains almost unchanged (ratio of A:B approximately 1:6 to 1:5) over this wide pH range. However, strong chemical exchange cross peaks appear in the nuclear Overhauser effect spectroscopy/exchange spectroscopy (NOESY/EXSY) spectra for the heme methyl resonances at low pH (pH* 4.0-5.5), which indicate chemical exchange between two species. We have shown these to be two different exogenous ImH or Hm orientations that are denoted B and B', with the ImH plane nearly parallel and perpendicular to the ImH plane of the protein-provided His57, respectively. The wt NP2-CN complex also shows EXSY cross peaks due to chemical exchange, which is shown to be a result of interchange between two ruffling distortions of the heme. The same ruffling distortion interchange is also responsible for the ImH and Hm chemical exchange. For the three NP2(V24E) ligand complexes, no EXSY cross peaks are observed, but the A:B ratios change dramatically with pH. The fact that heme favors the A orientation highly for NP2(V24E) at low pH as compared with wt NP2 is believed to be due to the steric effect of the V24E mutation. The existence of the B' species at lower pH for wt NP2 complexes and the increase in A heme orientation at lower pH for NP2(V24E) are believed to be a result of a change in structure near Glu53 when it is protonated at low pH. 1H{13C} heteronuclear multiple quantum coherence (HMQC) spectra are very helpful for the assignment of heme and nearby protein side chain resonances.

SUBMITTER: Yang F

PROVIDER: S-EPMC2847153 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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1H and 13C NMR spectroscopic studies of the ferriheme resonances of three low-spin complexes of wild-type nitrophorin 2 and nitrophorin 2(V24E) as a function of pH.

Yang Fei F Knipp Markus M Shokhireva Tatiana K TK Berry Robert E RE Zhang Hongjun H Walker F Ann FA

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 20090611 7

The ferriheme resonances of the low-spin (S = 1/2) complexes of wild-type (wt) nitrophorin 2 (NP2) and its heme pocket mutant NP2(V24E) with imidazole (ImH), histamine (Hm), and cyanide (CN(-)) as the sixth ligand have been investigated by NMR spectroscopy as a function of pH (4.0-7.5). For the three wt NP2 complexes, the ratio of the two possible heme orientational isomers, A and B, remains almost unchanged (ratio of A:B approximately 1:6 to 1:5) over this wide pH range. However, strong chemica ...[more]

PMID: 19517143

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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.

Project description:The measurement of long-range distances remains a challenge in solid-state NMR structure determination of biological macromolecules. In 2D and 3D correlation spectra of uniformly (13)C-labeled biomolecules, inter-residue, inter-segmental, and intermolecular (13)C-(13)C cross peaks that provide important long-range distance constraints for three-dimensional structures often overlap with short-range cross peaks that only reflect the covalent structure of the molecule. It is therefore desirable to develop new approaches to obtain spectra containing only long-range cross peaks. Here we show that a relaxation-compensated modification of the commonly used 2D (1)H-driven spin diffusion (PDSD) experiment allows the clean detection of such long-range cross peaks. By adding a z-filter to keep the total z-period of the experiment constant, we compensate for (13)C T1 relaxation. As a result, the difference spectrum between a long- and a scaled short-mixing time spectrum show only long-range correlation signals. We show that one- and two-bond cross peaks equalize within a few tens of milliseconds. Within ~200 ms, the intensity equilibrates within an amino acid residue and a monosaccharide to a value that reflects the number of spins in the local network. With T1 relaxation compensation, at longer mixing times, inter-residue and inter-segmental cross peaks increase in intensity whereas intra-segmental cross-peak intensities remain unchanged relative to each other and can all be subtracted out. Without relaxation compensation, the difference 2D spectra exhibit both negative and positive intensities due to heterogeneous T1 relaxation in most biomolecules, which can cause peak cancellation. We demonstrate this relaxation-compensated difference PDSD approach on amino acids, monosaccharides, a crystalline model peptide, a membrane-bound peptide and a plant cell wall sample. The resulting difference spectra yield clean multi-bond, inter-residue and intermolecular correlation peaks, which are often difficult to resolve in the parent 2D spectra.

Dataset Information

1H and 13C NMR spectroscopic studies of the ferriheme resonances of three low-spin complexes of wild-type nitrophorin 2 and nitrophorin 2(V24E) as a function of pH.

Publications

1H and 13C NMR spectroscopic studies of the ferriheme resonances of three low-spin complexes of wild-type nitrophorin 2 and nitrophorin 2(V24E) as a function of pH.

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