Narrow Results

A new total synthesis of a protoporphyrin IX derivative in which the α-methylene protons of the 13,17-(2-methoxycarbonylethyl) substituents are regioselectively deuterated is described. The deuterated porphyrin was obtained using the oxidative cyclization of an a,c-biladiene dihydrobromide.

Computational studies exploring the extent to which differences in proximal axial ligands modulate structure, spectra, and function of peroxidases have been performed. To this end, three heme models of compound I were characterized differing only in the axial ligand. The axial ligands considered were L=ImH, Im^-, that are alternative protonation models for a typical peroxidase with an imidazole ligand such as horseradish peroxidase (HRP-I), and L=SCH- that is a model for an unsual peroxidase, chloroperoxidase (CPO-I). Density functional calculations (DFTs) were performed to determine the optimized geometries and electronic structure of each of these three species. Their electronic spectra were also calculated at the DFT optimized geometries, using the INDO/S/CI method. The results of these studies led to the following conclusions: (1) the presence of the nearby Asp in a typical peroxidase does indeed decrease the energy required to deprotonate the imidazole making the two forms essentially degenerate, (2) neither the state of protonation of the imidazole ligand nor the change in axial ligand from an imidazole in typical peroxidases such as HRP to a mercaptide in CPO significantly alters the characteristics of the lowest energy spin state or the electronic structure of compound I in a way that can obviously affect function, (3) both the Im^- and ImH forms of the peroxidase compound I (HRP-I) lead to the same dramatic reduction in intensity relative to the ferric resting form observed experimentally. However, only in the ImH form of HRP-I does the calculated relative shift of one component of the Soret bands relative to CPO-I agree with that observed in the transient spectra of HRP-I compared to CPO-I. These results taken together strongly indicate that factors other than the nature of the proximal axial ligand are the main determinants of function.

Non-local Density Functional Theory (DFT) is applied to the calculation of geometry and vibrational frequencies of Fe^II(porphine)(imidazole)(CO), a model for CO adducts of heme proteins. Bond distances and angles are in agreement with crystallographic data, and frequencies are correctly calculated for C–O and Fe–C stretching and for Fe–C-O bending. This last mode is actually the out-of-phase combination of Fe–C–O bending and Fe–C tilting coordinates, which are heavily mixed because of a large bend–tilt interaction force constant. The in-phase combination is predicted at a very low frequency, 73 cm^-1, and to have a low infrared intensity; attempts to detect it in far-IR spectra via ¹³C¹⁸O isotope sensitivity have been unsuccessful. The stretch–bend interaction lowers the energy required for FeCO distortion. A soft potential may account for the wide range of crystallographically determined Fe–C–O displacements and orientations in myoglobin (Mb). The minimum energy path for displacement of the O atom from the heme normal was calculated by relaxing the structure while constraining only the O atom displacement from the heme normal. Energies of 0.2 to 3.5 kcal mol^-1 are required for the range of reported displacement, 0.3–1.3 Å. However, vibrational spectroscopy limits the allowable displacement to the low end of this range. The O atom displacement is computed via DFT to be 0.6 Å for a 7 ° angle of the C–O stretching IR dipole relative to the heme normal, the maximum value compatible with IR polarization measurements on MbCO. FeCO distortion is predicted to diminish both ν_CO and ν_FeC, thereby producing deviations from the well-established backbonding correlation; the scatter of the data permits a maximum displacement of 0.5 Å. This displacement would cost about 1.6 kcal mol^-1 of steric energy. A small distortion energy is consistent with the CO affinity changes produced by mutations of the distal histidine residue in Mb. Taking the leucine mutant as reference, we estimate the 1.6 kcal mol^-1 affinity loss in the wild-type protein to be the resultant of a 0.0–1.6 kcal steric inhibition, a 0.5 kcal mol^-1 attraction of the distal histidine sidechain for the bound CO [weak H-bond], and a 0.5–2.1 kcal mol^-1 attraction of the same side-chain for a water molecule in the deoxy protein. The observed 2.3 kcal mol^-1O₂ affinity increase in the wild-type protein relative to the leucine mutant then implies a 2.8–4.4 kcal mol^-1 attraction of the histidine sidechain for bound O₂, consistent with a substantial H-bond interaction with the distal histidine. Thus steric inhibition can account for only a minor fraction of the discrimination factor against CO and in favor of O₂ which is produced by the heme–myoglobin interaction.

The main topics in resonance Raman spectroscopy presented at ICPP-2 in Kyoto are briefly discussed. These include: (i) coherent spectroscopy and low frequency vibrations of ligand-photodissociated heme proteins, (ii) vibrational relaxation revealed by time-resolved anti-Stokes Raman spectroscopy, (iii) electron transfer in porphyrin arrays, (iv) vibrational assignments of tetraazaporphyrins and (v) resonance Raman spectra of an NO storing protein, nitrophorin.

This paper covers a detailed analysis of the coordination changes taking place at the active sites in both Cu and Ni reconstituted hemoglobin as a function of pH. These experiments provide insight into how proteins are held in their native configuration. The EPR results of CuHb reveal that the species formed in extreme acidic condition were different from those formed at extreme basic condition. At pH 3 we see an isotropic spectrum characteristic of 4-coordinated species, while at pH 12 there is an indication of equilibrium between mixtures of species. Further support for the above coordination changes is obtained from FT-Raman of NiHb at different pH conditions. At pH 3 all the 5-coordination marker bands are lost and there is a shift in the 4-coordination marker band, while at pH 12 both 4- and 5-coordination marker bands are still seen with slight shift in their positions. In addition to this, we could see a new peak at 1633 cm⁻¹. The coordination changes as a function of pH could be seen for both CuHb and NiHb using UV-visible spectroscopic techniques.

The spectroscopic characterization of peroxo- and hydroperoxo- intermediates of heme proteins and enzymes has long interested scientists studying the structure and function of these important biochemical systems. Until very recently, little progress had been made in studying these fleeting intermediates by vibrational spectroscopic methods. In this brief review, recent studies reporting the Resonance Raman and Infrared spectra of such intermediates and pertinent model compounds are reviewed and compared to corresponding studies utilizing electronic absorption and electron paramagnetic resonance spectrometric methods.

Staphylococcus aureus is a human pathogen that results in numerous infections in hospital settings and recently also in the wider community. Its antibiotic resistant forms are causing considerable alarm. A series of surface-anchored proteins that have heme uptake and transport properties have been reported. Through the use of absorption and magnetic circular dichroism spectroscopies and mass spectrometry, the iron-free, protoporphyrin IX and the iron-containing, heme-binding characteristics of bacterial rIsdC have been obtained. Mass spectrometry showed that following isolation and purification, the rIsdC is bound predominantly to protoporphyrin IX and to a lesser extent heme, unlike the case of rIsdA, which binds predominantly heme. Magnetic circular dichroism analysis provided further information regarding porphyrin binding because the characteristic magnetic circular dichroism band envelopes for the iron-free protoporphyrin IX and the iron-containing heme can be clearly distinguished in the spectrum of the rIsdC. Analysis of these spectral data showed that the minor heme component exists as a high-intermediate spin state ferric heme when bound to rIsdC, similar to the high-spin ferric heme reported for the rIsdA protein.