Jeffrey S. de Ropp

Axial histidyl imidazole non-exchangeable proton resonances as indicators of imidazole hydrogen bonding in ferric cyanide complexes of heme peroxidases.

Proton NMR spectra of a model of low-spin cyanide complexes of ferric hemoproteins indicate that two broad single-protein resonances from the axial imidazole can be resolved outside the diamagnetic spectral region. Upon deprotonation of the imidazole in the model, the upfield resonance shifts dramatically to higher field, suggesting that its position may reflect the degree of hydrogen bonding or proton donation of the imidazole. Met-cyano myoglobin reveals a pair of such broad peaks in the regions expected for an essentially neutral axial imidazole. In the cyano complexes of horseradish peroxidase and cytochrome c peroxidase, a pair of single-proton resonances are located which are assigned to the same imidazole protons on the basis of their linewidth and shift changes upon altering the heme substituents. The upfiled proton, however, is found at much higher field than in metMbCN. The upfield bias of this resonance is taken as evidence for appreciable imidazolate character for the axial ligand in these heme peroxidases.

Automated screening for metabolites in complex mixtures using 2D COSY NMR spectroscopy

One of the greatest challenges in metabolomics is the rapid and unambiguous identification and quantification of metabolites in a biological sample. Although one-dimensional (1D) proton nuclear magnetic resonance (NMR) spectra can be acquired rapidly, they are complicated by severe peak overlap that can significantly hinder the automated identification and quantification of metabolites. Furthermore, it is currently not reasonable to assume that NMR spectra of pure metabolites are available a priori for every metabolite in a biological sample. In this paper we develop and report on tests of methods that assist in the automatic identification of metabolites using proton two-dimensional (2D) correlation spectroscopy (COSY) NMR. Given a database of 2D COSY spectra for the metabolites of interest, our methods provide a list sorted by a heuristic likelihood of the metabolites present in a sample that has been analyzed using 2D COSY NMR. Our models attempt to correct the displacement of the peaks that can occur from one sample to the next, due to pH, temperature and matrix effects, using a statistical and chemical model. The correction of one peak can result in an implied correction of others due to spin–spin coupling. Furthermore, these displacements are not independent: they depend on the relative position of functional groups in the molecule. We report experimental results using defined mixtures of amino acids as well as real complex biological samples that demonstrate that our methods can be very effective at automatically and rapidly identifying metabolites.

NMR Methodology for Paramagnetic Proteins

While it has long been recognized that both the hyperfine shifts and paramagnetic relaxation can provide unique and valuable information on the electronic, magnetic, and molecular structural properties of the active site of paramagnetic metalloproteins, access to this information has been thwarted by the absence of a systematic and reliable strategy for assigning the resonances to specific nuclei in the molecule. Thus the initial excitement about and interest in NMR spectroscopy of paramagnetic proteins in the late 60s to middle 70s faded somewhat on the full realization of the apparent barriers to unambiguous signal assignment. The early strategies for assigning hyperfine-shifted resonances in a paramagnetic protein relied on a number of methods, of which most had only a very limited applicability. On the one hand was the least definitive method, namely, the comparison of the shifts of model compounds with those of intact proteins; the selection of appropriate models often relied heavily on intuition (La Mar, 1979). While immensely useful as a starting point in the absence of other data, this method is unreliable except as a guide for the design of more definitive experiments. On the other extreme was the very direct approach represented by the specific isotope labeling of individual functional groups of native prosthetic groups. This, however, involves laborious synthetic methodology and is restricted to the class of b-type hemoproteins where the reversibly removable heme could be replaced by one with only 2H for 1H or 13C for 12C substitution at restricted positions (Mayer et al., 1974; La Mar et al., 1978; La Mar et al., 1980a, b, 1981a, b, 1985, 1988; Satterlee et al., 1983; de Ropp et al., 1984; Sankar et al., 1987). Both of the above strategies, moreover, are restricted by their nature to identifying signals solely from residues directly coordinated to the paramagnetic metal ion.

2D NMR of paramagnetic metalloenzymes: Cyanide-inhibited horseradish peroxidase

SummaryTwo-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 ∼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 cytochromec 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 ∼ 100 Hz. Moreover, paramagnetic-induced hyperfine shifts and linewidths to ∼100 Hz need not interfere with the complete solution structure determination of a small paramagnetic protein solely on the basis of 2D NMR data.

Assignment of exchangeable proximal histidine resonances in high-spin ferric hemoproteins: substrate binding in horseradish peroxidase.

Abstract Single-proton, exchangeable resonances have been detected in the high spin ferric hemoproteins, met-aquo myoglobin and horseradish peroxidase, which can be assigned to the proximal histidyl imidazole by virtue of their very large hyperfine shifts. While this proton is relatively labile in myoglobin, it is exchangeable in HRP only at extreme pH values, indicating a buried heme pocket. The insensitivity of the imidazole peak of HRP to substrate binding argues against direct interaction of imidazole and substrate.

Solvent isotope effects on NMR spectral parameters in high-spin ferric hemoproteins: an indirect probe for distal hydrogen bonding.

The influence of solvent isotope composition on 1H-NMR resonance position and linewidth of heme methyls has been investigated for a variety of high-spin ferric hemoproteins for the purpose of detecting hydrogen-bonding interactions in the heme cavity. Consistently larger hyperfine shifts and paramagnetic linewidths in 2H2O than 1H2O are observed for metmyoglobins and methemoglobin possessing a coordinated water molecule. The analysis of the dynamics of labile proton exchange in sperm whale metmyoglobin, and the absence of any isotope effects in the five-coordinate Aplysia metmyoglobin, indicate that the significant axial modulation of heme electronic structure by solvent isotope is consistent with arising from distal hydrogen-bonding interactions. The presence or absence of similarly large isotope effects on shifts and linewidths in other hemoproteins, depending on the presence of a bound water in the distal heme pocket, suggests that this isotope effect can serve as a probe for the presence of such bound water. The absence of any detectable isotope effect on either shifts or linewidths in resting-state horseradish peroxidase supports a five-coordinate structure with bound water absent from the vicinity of the iron.

Electrophilic deuteriation of natural porphyrin derivatives

The proportion of electrophilic deuteriation at individual meso positions in unsymmetrically substituted porphyrins depends upon the nature of the substituents on the adjacent pyrrolic subunits; in the case of protoporphyrin-IX, these observations allow the first unequivocal assignment of all four meso protons in the n.m.r. spectrum of dicyanoferriprotoheme.

Assignment Strategies and Structure Determination in Cyanide-Inhibited Heme Peroxidases

A strategy is described for locating and assigning all of the hyperfine-shifted and/or relaxed resonances in the active site of the low-spin, cyanide-inhibited complex of horseradish peroxidase. The serious problems in spectral resolution and dynamic range due to the large size (44 kDa) of the protein can be overcome by taking advantage of the strong temperature dependence of the hyperfine shift, and by use of DEFT or WEFT pulse sequences in conjunction with standard 2D experiments. Detailed analysis of COSY experiments reveals that cross-correlation contributes significantly to cross peak intensity and dominates for geminal protons. It is shown that many COSY cross peaks, in fact, are inter-residue cross correlation peaks. Hence COSY has limited use in mapping scalar connectivities, even in its phase-sensitive form. TOCSY, on the other hand, particularly when ROESY is suppressed, provides an effective method for mapping essentially all spin systems. The sequence-specific assignment of active site residues via standard backbone NOES Y connectivities in 2H2O is greatly facilitated by the very slow exchange rates of the peptide protons for both the proximal and distal helices. The combination of 2D NMR methods lead to the complete assignment of the active site protons <6.5 A from the iron. The resulting dipolar shifts for non-coordinated residues are shown to be quantitatively described by the orientation of the magnetic axes which reflect a tilt of the Fe-CN unit away from the heme normal. It is likely that similar methods will allow detailed structural studies in a variety of comparably sized cyanide-inhibited heme peroxidases.

The State of Protonation of the Proximal Histidyl Imidazole in Horseradish Peroxidase

The ease of formation of iron(IV) complexes in horseradish peroxidase as well as in other hemoproteins has been cited to support the view that in these proteins the proximal histidyl imidazole is deprotonated. However, most of the supporting evidence for this proposal has been indirect. From a detailed comparison of the proton nmr spectra of horseradish peroxidase in several oxidation/spin/ligation states with other hemoproteins and model compounds, it is shown hereby direct detection of the N1H signal, that the proximal histidyl imidazole in horseradish peroxidase is definitely not deprotonated, but most likely involved in hydrogen bonding interactions with the protein in at least the reduced and the cyanide-ligated states.