Human serum albumin (HSA), the most prominent protein in blood plasma, is able to bind a wide range of endogenous and exogenous compounds. Among the endogenous ligands, HSA is a significant transporter of heme, the heme-HSA complex being present in blood plasma. Drug binding to heme-HSA affects allosterically the heme affinity for HSA and vice versa. Heme-HSA, heme, and their complexes with ibuprofen have been characterized by electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy. Comparison of the results for the heme and heme-HSA systems has provided insight into the structural consequences on the heme pocket of ibuprofen binding. The pentacoordinate tyrosine-bound heme coordination of heme-HSA, observed in the absence of ibuprofen, becomes hexacoordinate low spin upon ibuprofen binding, and heme dissociates at increasing drug levels. The electronic absorption spectrum and nu(Fe-CO)/nu(CO) vibrational frequencies of the CO-heme-HSA-ibuprofen complex, together with the observation of a Fe-His Raman mode at 218 cm(-1) upon photolysis of the CO complex and the low spin EPR g values indicate that a His residue is one of the low spin axial ligands, the sixth ligand probably being Tyr161. The only His residue in the vicinity of the heme Fe atom is His146, 9 A distant in the absence of the drug. This indicates that drug binding to heme-HSA results in a significant rearrangement of the heme pocket, implying that the conformational adaptability of HSA involves more than the immediate vicinity of the drug binding site. As a whole, the present spectroscopic investigation supports the notion that HSA could be considered as the prototype of monomeric allosteric proteins.

Magnetic nanoparticles, MNPs, mineralized within a human ferritin protein cage, HFt, can represent an appealing platform to realize smart therapeutic agents for cancer treatment by drug delivery and magnetic fluid hyperthermia, MFH. However, the constraint imposed by the inner diameter of the protein shell (ca. 8 nm) prevents its use as heat mediator in MFH when the MNPs comprise pure iron oxide. In this contribution, we demonstrate how this limitation can be overcome through the controlled doping of the core with small amount of Co(II). Highly monodisperse doped iron oxide NPs with average size of 7 nm are mineralized inside a genetically modified variant of HFt, carrying several copies of α-melanocyte-stimulating hormone peptide, which has already been demonstrated to have excellent targeting properties toward melanoma cells. HFt is also conjugated to poly(ethylene glycol) molecules to increase its in vivo stability. The investigation of hyperthermic properties of HFt-NPs shows that a Co doping of 5% is enough to strongly enhance the magnetic anisotropy and thus the hyperthermic efficiency with respect to the undoped sample. In vitro tests performed on B16 melanoma cell line demonstrate a strong reduction of the cell viability after treatment with Co doped HFt-NPs and exposure to the alternating magnetic field. Clear indications of an advanced stage of apoptotic process is also observed from immunocytochemistry analysis. The obtained data suggest this system represents a promising candidate for the development of a protein-based theranostic nanoplatform.

Variants of the copper-containing nitrite reductase (NiR) of Alcaligenes faecalis S6 were constructed by site-directed mutagenesis, by which the C-terminal histidine ligand (His145) of the Cu in the type-1 site was replaced by an alanine or a glycine. The type-1 sites in the NiR variants as isolated, are in the reduced form, but can be oxidized in the presence of external ligands, like (substituted) imidazoles and chloride. The reduction potential of the type-1 site of NiR-H145A reconstituted with imidazole amounts to 505 mV vs NHE (20 degrees C, pH 7, 10 mM imidazole), while for the native type-1 site it amounts to 260 mV. XRD data on crystals of the reduced and oxidized NiR-H145A variant show that in the reduced type-1 site the metal is 3-coordinated, but in the oxidized form takes up a ligand from the solution. With the fourth (exogenous) ligand in place the type-1 site is able to accept electrons at about the same rate as the wt NiR, but it is unable to pass the electron onto the type-2 site, leading to loss of enzymatic activity. It is argued that the uptake of an electron by the mutated type-1 site is accompanied by a loss of the exogenous ligand and a concomitant rise of the redox potential. This rise effectively traps the electron in the type-1 site.

Doping of biocompatible nanomaterials with magnetic phases is currently one of the most promising strategies for the development of advanced magnetic biomaterials. However, especially in the case of iron-doped magnetic hydroxyapatites, it is not clear if the magnetic features come merely from the magnetic phases/ions used as dopants or from complex mechanisms involving interactions at the nanoscale. Here, we report an extensive chemical-physical and magnetic investigation of three hydroxyapatite nanocrystals doped with different iron species and containing small or no amounts of maghemite as a secondary phase. The association of several investigation techniques such as X-ray absorption spectroscopy, Mössbauer, magnetometry, and TEM allowed us to determine that the unusual magnetic properties of Fe-doped hydroxyapatites (FeHA) occur by a synergy of two different phenomena: i.e., (i) interacting superparamagnetism due to the interplay between iron-doped apatite and iron oxide nanoparticles as well as to the occurrence of dipolar interactions and (ii) interacting paramagnetism due to Fe ions present in the superficial hydrated layer of the apatite nanophase and, to a lesser extent, paramagnetism due to isolated Fe ions in the apatite lattice. We also show that a major player in the activation of the above phenomena is the oxidation of Fe into Fe, as induced by the synthesis process, and their consequent specific positioning in the FeHA structure.

In spite of the tremendous progress in the field of pulse electron paramagnetic resonance (EPR) in recent years, these techniques have been scarcely used to investigate high-spin (HS) ferric heme proteins. Several technical and spin-system-specific reasons can be identified for this. Additional problems arise when no single crystals of the heme protein are available. In this work, we use the example of a frozen solution of aquometmyoglobin (metMb) to show how a multi-frequency pulse EPR approach can overcome these problems. In particular, the performance of the following pulse EPR techniques are tested: Davies electron nuclear double resonance (ENDOR), hyperfine correlated ENDOR (HYEND), electron-electron double resonance (ELDOR)-detected NMR, and several variants of hyperfine sublevel correlation (HYSCORE) spectroscopy including matched and SMART HYSCORE. The pulse EPR experiments are performed at X-, Q- and W-band microwave frequencies. The advantages and drawbacks of the different methods are discussed in relation to the nuclear interaction that they intend to reveal. The analysis of the spectra is supported by several simulation procedures, which are discussed. This work focuses on the analysis of the hyperfine and nuclear-quadrupole tensors of the strongly coupled nuclei of the first coordination sphere, namely, the directly coordinating heme and histidine nitrogens and the 17O nucleus of the distal water ligand. For the latter, 17O-isotope labeling was used. The accuracy of our results and the spectral resolution are compared in detail to an earlier single-crystal continuous-wave ENDOR study on metMb, and it will be shown how additional information can be obtained from the multi-frequency approach. The current work is therefore prone to become a template for future EPR/ENDOR investigations of HS ferric heme proteins for which no single crystals are available.

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The interaction between cytochrome c (Cyt c) and cardiolipin (CL) plays a vital role in the early stages of apoptosis. The binding of CL to Cyt c induces a considerable increase in its peroxidase activity that has been attributed to the partial unfolding of the protein, dissociation of the Met80 axial ligand, and formation of non-native conformers. Although the interaction between Cyt c and CL has been extensively studied, there is still no consensus regarding the conformational rearrangements of Cyt c that follow the protein-lipid interaction. To rationalize the different results and gain better insight into the Cyt c-CL interaction, we have studied the formation of the CL complex of the horse heart wild-type protein and selected mutants in which residues considered to play a key role in the interaction with CL (His26, His33, Lys72, Lys73, and Lys79) have been mutated. The analysis was conducted at both room temperature and low temperatures via ultraviolet-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. The trigger and the sequence of CL-induced structural variations are discussed in terms of disruption of the His26-Pro44 hydrogen bond. We unequivocally identify the sixth ligand in the partially unfolded, non-native low-spin state that Cyt c can adopt following the protein-lipid interaction, as a His ligation, ruling out the previously proposed involvement of a Lys residue or an OH ion.

In this work, the high-spin ferric form of the E7Q mutant of human neuroglobin (E7Q-NGB) is studied by X-band continuous-wave electron paramagnetic resonance (CW EPR) and hyperŸ237 sublevel correlation (HYSCORE) spectroscopy. It is shown that the use of matched pulses in the HYSCORE experiment is essential to observe the nitrogen speetral contributions. The validity of approximating the high-spin Fe(IID system (S = 5/2) as an effective S = 1/2 system is tested and the consequences for the HYSCORE simulations are highlighted. Comparative HYSCORE experiments combined with deuterium exchange experiments for aquometmyoglobin and fer¡ E7Q-NGB clearly show that the heme iron of the latter protein is pentacoordinated, tacking the distal water. Furthermore, CW EPR experiments show that, at high pH, the E10K residue is coordinating to the heme iron in this globin. These observations ate corroborated by resonance Raman experiments and could also be reproduced for other E7 mutants of human and mouse neuroglobin. Finally, the proton and nitrogen hyperfine and nuclear quadrupole parameters obtained for ferric E7Q-NGB are discussed in detail.