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This review provides examples illustrating the powerful combination of resonance Raman spectroscopy and site-directed mutagenesis to investigate the structure-function relationship in structurally different heme proteins with diverse physiological functionality. The selective mutation of key amino acid residues gives rise to distinct spectroscopic fingerprints, as a result of the subtle alterations of the heme pocket environment. This review includes, but it is not limited to, the study of: i) the interactions between bound exogenous ligands with distal residues, ii) the effects of hydrogen bonds between the proximal residues and the surrounding cavity, iii) the interaction between the peripheral substituents of the heme group with the protein matrix with the concomitant effect on specific biological processes.

We produced a neuroglobin variant, namely, Ngb CDless, with the excised CDloop- and D-helix, directly joining the C- and E-helices. The CDless variant retained bis-His hexacoordination, and we investigated the role of the CDloop-D-helix unit in controlling the CO binding and structural dynamics by an integrative approach based on X-ray crystallography, rapid mixing, laser flash photolysis, resonance Raman spectroscopy, and molecular dynamics simulations. Rapid mixing and laser flash photolysis showed that ligand affinity was unchanged with respect to the wild-type protein, albeit with increased on and off constants for rate-limiting heme iron hexacoordination by the distal His64. Accordingly, resonance Raman spectroscopy highlighted a more open distal pocket in the CO complex that, in agreement with MD simulations, likely involves His64 swinging inward and outward of the distal heme pocket. Ngb CDless displays a more rigid overall structure with respect to the wild type, abolishing the structural dynamics of the CDloop-D-helix hypothesized to mediate its signaling role, and it retains ligand binding control by distal His64. In conclusion, this mutant may represent a tool to investigate the involvement of CDloop-D-helix in neuroprotective signaling in a cellular or animal model.

Coproporphyrin ferrochelatases (CpfCs) are enzymes catalyzing the penultimate step in the coproporphyrin-dependent (CPD) heme biosynthesis pathway, which is mainly utilized by monoderm bacteria. Ferrochelatases insert ferrous iron into a porphyrin macrocycle and have been studied for many decades, nevertheless many mechanistic questions remain unanswered to date. Especially CpfCs, which are found in the CPD pathway, are currently in the spotlight of research. This pathway was identified in 2015 and revealed that the correct substrate for these ferrochelatases is coproporphyrin III (cpIII) instead of protoporphyrin IX, as believed prior the discovery of the CPD pathway. The chemistry of cpIII, which has four propionates, differs significantly from protoporphyrin IX, which features two propionate and two vinyl groups. These findings let us to thoroughly describe the physiological cpIII-ferrochelatase complex in solution and in the crystal phase. Here, we present the first crystallographic structure of the CpfC from the representative monoderm pathogen Listeria monocytogenes bound to its physiological substrate, cpIII, together with the in-solution data obtained by resonance Raman and UV-vis spectroscopy, for wild-type ferrochelatase and variants, analyzing propionate interactions. The results allow us to evaluate the porphyrin distortion and provide an in-depth characterization of the catalytically-relevant binding mode of cpIII prior to iron insertion. Our findings are discussed in the light of the observed structural restraints and necessities for this porphyrin-enzyme complex to catalyze the iron insertion process. Knowledge about this initial situation is essential for understanding the preconditions for iron insertion in CpfCs and builds the basis for future studies.

Coproporpyhrin III is the substrate of coproporphyrin ferrochelatases (CpfCs). These enzymes catalyse the insertion of ferrous iron into the porphyrin ring. This is the penultimate step within the coproporphyrin-dependent haeme biosynthesis pathway. This pathway was discovered in 2015 and is mainly utilised by monoderm bacteria. Prior to this discovery, monoderm bacteria were believed to utilise the protoporphyrin-dependent pathway, analogously to diderm bacteria, where the substrate for the respective ferrochelatase is protoporphyrin IX, which has two propionate groups at positions 6 and 7 and two vinyl groups at positions 2 and 4. In this work, we describe for the first time the interactions of the four-propionate substrate, coproporphyrin III, and the four-propionate product, iron coproporphyrin III (coproheme), with the CpfC from Listeria monocytogenes and pin down differences with respect to the protoporphyrin IX and haeme b complexes in the wild-type (WT) enzyme. We further created seven LmCpfC variants aiming at altering substrate and product coordination. The WT enzyme and all the variants were comparatively studied by spectroscopic, thermodynamic and kinetic means to investigate in detail the H-bonding interactions, which govern complex stability and substrate specificity. We identified a tyrosine residue (Y124 in LmCpfC), coordinating the propionate at position 2, which is conserved in monoderm CpfCs, to be highly important for binding and stabilisation. Importantly, we also describe a tyrosine-serine-threonine triad, which coordinates the propionate at position 4. The study of the triad variants indicates structural differences between the coproporphyrin III and the coproheme complexes. ENZYME: EC 4.99.1.9.

Nitrobindins (Nbs) are all-β-barrel heme-proteins present in all the living kingdoms. Nbs inactivate reactive nitrogen species by sequestering NO, converting NO to HNO

Nitrobindins (Nbs) are all-β-barrel heme-proteins present in prokaryotes and eukaryotes. Although the physiological role(s) of Nbs are still unclear, it has been postulated that they are involved in the NO/O2 metabolism, which is particularly relevant in fishes for the oxygen supply. Here, the reactivity of ferrous Danio rerio Nb (Dr-Nb(II)) towards NO has been investigated from the spectroscopic and kinetic viewpoints and compared with those of Mycobacterium tuberculosis Nb, Arabidopsis thaliana Nb, Homo sapiens Nb, and Equus ferus caballus myoglobin. Between pH 5.5 and 9.1 at 22.0 °C, Dr-Nb(II) nitrosylation is a monophasic process; values of the second-order rate constant for Dr-Nb(II) nitrosylation and of the first-order rate constant for Dr-Nb(II)-NO denitrosylation are pH-independent ranging between 1.6 × 106 M-1 s-1 and 2.3 × 106 M-1 s-1 and between 5.3 × 10-2 s-1 and 8.2 × 10-2 s-1, respectively. Interestingly, both UV-Vis and EPR spectroscopies indicate that the heme-Fe(II) atom of Dr-Nb(II)-NO is five-coordinated. Kinetics of Dr-Nb(II) nitrosylation may reflect the ligand accessibility to the metal center, which is likely impaired by the crowded network of water molecules which shields the heme pocket from the bulk solvent. On the other hand, kinetics of Dr-Nb(II)-NO denitrosylation may reflect an easy pathway for the ligand escape into the outer solvent.

Coproheme decarboxylases (ChdCs) are utilized by monoderm bacteria to produce heme b by a stepwise oxidative decarboxylation of the 2- and 4-propionate groups of iron coproporphyrin III (coproheme) to vinyl groups. This work compares the effect of hemin reconstitution versus the hydrogen peroxide-mediated conversion of coproheme to heme b in the actinobacterial ChdC from Corynebacterium diphtheriae (CdChdC) and selected variants. Both ferric and ferrous forms of wild-type (WT) CdChdC and its H118A, H118F, and A207E variants were characterized by resonance Raman and UV-vis spectroscopies. The heme b ligand assumes the same conformation in the WT active site for both the reconstituted and H

The actinobacterial coproheme decarboxylase from

In the present review, examples are provided illustrating the application of resonance Raman microscopy to heme protein single crystals to highlight the artifacts induced by the crystallization process or the conformational alteration induced by cooling. Moreover, the structural information determined from the RR spectra of heme proteins in solution and crystals is compared to that obtained from their X-ray structures to show how the combined spectroscopic/crystallographic approach is a powerful weapon in the structural biologist’s armamentarium.

Monoderm bacteria utilize coproheme decarboxylases (ChdCs) to generate heme b by a stepwise decarboxylation of two propionate groups of iron coproporphyrin III (coproheme), forming two vinyl groups. This work focuses on actinobacterial ChdC from Corynebacterium diphtheriae (CdChdC) to elucidate the hydrogen peroxide-mediated decarboxylation of coproheme via monovinyl monopropionyl deuteroheme (MMD) to heme b, with the principal aim being to understand the reorientation mechanism of MMD during turnover. Wild-type CdChdC and variants, namely H118A, H118F, and A207E, were studied by resonance Raman and ultraviolet-visible spectroscopy, mass spectrometry, and molecular dynamics simulations. As actinobacterial ChdCs use a histidine (H118) as a distal base, we studied the H118A and H118F variants to elucidate the effect of 1) the elimination of the proton acceptor and 2) steric constraints within the active site. The A207E variant mimics the proximal H-bonding network found in chlorite dismutases. This mutation potentially increases the rigidity of the proximal site and might impair the rotation of the reaction intermediate MMD. We found that both wild-type CdChdC and the variant H118A convert coproheme mainly to heme b upon titration with H2O2. Interestingly, the variant A207E mostly accumulates MMD along with small amounts of heme b, whereas H118F is unable to produce heme b and accumulates only MMD. Together with molecular dynamics simulations, the spectroscopic data provide insight into the reaction mechanism and the mode of reorientation of MMD, i.e., a rotation in the active site versus a release and rebinding.

13 pags., 6 figs., 1 tab. -- This article belongs to the Special Issue Nanomaterials in Surface-Enhanced Raman Spectroscopy, Silver nanoparticles functionalized with thiolated β-cyclodextrin (CD-SH) were employed for the detection of bisphenols (BPs) A, B, and S by means of surface-enhanced Raman spectroscopy (SERS). The functionalization of Ag nanoparticles with CD-SH leads to an improvement of the sensitivity of the implemented SERS nanosensor. Using a multivariate analysis of the SERS data, the limit of detection of these compounds was estimated at about 10−7 M, in the range of the tens of ppb. Structural analysis of the CD-SH/BP complex was performed by density functional theory (DFT) calculations. Theoretical results allowed the assignment of key structural vibrational bands related to ring breathing motions and the inter-ring vibrations and pointed out an external interaction due to four hydrogen bonds between the hydroxyl groups of BP and CD located at the external top of the CD cone. DFT calculations allowed also checking the interaction energies of the different molecular species on the Ag surface and testing the effect of the presence of CD-SH on the BPs’ affinity. These findings were in agreement with the experimental evidences that there is not an actual inclusion of BP inside the CD cavity. The SERS sensor and the analysis procedure of data based on partial least square regression proposed here were tested in a real sample consisting of the detection of BPs in milk extracts to validate the detection performance of the SERS sensor., This research was supported by the Italian Ministry of Health, Rome, Italy (IZSLT11/17 RC) (G.S., B.N., and C.F.), MINECO/FEDER (FIS2017-84318-R) (S.S.C.), and by Junta de Andalucía/FEDER (UMA18-FEDERJA-049 and P18-RT-4592) (J.C.O.).

1 Pag., In the original article, there were three errors. The concentrations of antibiotics should be expressed in micrograms per milliliter; the pH of the Tris buffer should be 8.0; and the concentration of deoxyferrous globin should be stated as 2.5 micromolar. Two corrections have been made to: MATERIALS AND METHODS, Production and Purification of Recombinant Globins, paragraph 1, line 7 and line 17, “…of LB medium with 100 μg ml−1 ampicillin or 100 μg ml−1 kanamycin,…” “..20 mM Tris (pH 8.0) with 150 mM NaCl…” One correction has been made to: MATERIALS AND METHODS, Nitrite Reductase Activity, paragraph 1, line 4, “…were 2.5 μM and 0.05–1 mM, respectively.” The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

Different murine neuroglobin variants showing structural and dynamic alterations that are associated with perturbation of ligand binding have been studied: the CD loop mutants characterized by an enhanced flexibility (Gly-loop40-48 and Gly-loop44-47 ), the F106A mutant, and the double Gly-loop44-47 /F106A mutant. Their ferric resonance Raman spectra in solution and in crystals are almost identical. In the high-frequency region, the identification of a double set of core size marker bands indicates the presence of two 6-coordinate low spin species. The resonance Raman data, together with the corresponding crystal structures, indicate the presence of two neuroglobin conformers with a reversed (A conformer) or a canonical (B conformer) heme insertion orientation. With the identification of the marker bands corresponding to each conformer, the data indicate that the B conformer increases at the expense of the A form, predominantly in the Gly-loop44-47 /F106A double mutant, as confirmed by X-ray crystallography. This is the first time that a reversed heme insertion has been identified by resonance Raman in a native 6-coordinate low-spin heme protein. This diagnostic tool could be extended to other heme proteins in order to detect heme orientational disorder, which are likely to be correlated to functionally relevant heme dynamics. DATABASE: Crystallographic structure: structural data are deposited in the Protein Data Bank under the 6RA6 PDB entry.

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.

The heme enzyme chlorite dismutase (Cld) catalyzes the degradation of chlorite to chloride and dioxygen. Although structure and steady-state kinetics of Clds have been elucidated, many questions remain (e.g., the mechanism of chlorite cleavage and the pH dependence of the reaction). Here, we present high-resolution X-ray crystal structures of a dimeric Cld at pH 6.5 and 8.5, its fluoride and isothiocyanate complexes and the neutron structure at pH 9.0 together with the pH dependence of the Fe(III)/Fe(II) couple, and the UV-vis and resonance Raman spectral features. We demonstrate that the distal Arg127 cannot act as proton acceptor and is fully ionized even at pH 9.0 ruling out its proposed role in dictating the pH dependence of chlorite degradation. Stopped-flow studies show that (i) Compound I and hypochlorite do not recombine and (ii) Compound II is the immediately formed redox intermediate that dominates during turnover. Homolytic cleavage of chlorite is proposed.

The interaction between gold sub-nanometer clusters composed of ten atoms (Au10) and tetrakis(4-sulfonatophenyl)porphyrin (TPPS) was investigated through various spectroscopic techniques. Under mild acidic conditions, the formation, in aqueous solutions, of nanohybrid assemblies of porphyrin J-aggregates and Au10 cluster nanoparticles was observed. This supramolecular system tends to spontaneously cover glass substrates with a co-deposit of gold nanoclusters and porphyrin nanoaggregates, which exhibit circular dichroism (CD) spectra reflecting the enantiomorphism of histidine used as capping and reducing agent. The morphology of nanohybrid assemblies onto a glass surface was revealed by atomic force microscopy (AFM), and showed the concomitant presence of gold nanoparticles with an average size of 130 nm and porphyrin J-aggregates with lengths spanning from 100 to 1000 nm. Surface-enhanced Raman scattering (SERS) was observed for the nanohybrid assemblies.

Coproheme decarboxylase (ChdC) catalyzes the last step in the heme biosynthesis pathway of monoderm bacteria with coproheme acting both as redox cofactor and substrate. Hydrogen peroxide mediates the stepwise decarboxylation of propionates 2 and 4 of coproheme. Here we present the crystal structures of coproheme-loaded ChdC from Listeria monocytogenes (LmChdC) and the three-propionate intermediate, for which the propionate at position 2 (p2) has been converted to a vinyl group and is rotated by 90° compared to the coproheme complex structure. Single, double, and triple mutants of LmChdC, in which H-bonding interactions to propionates 2, 4, 6, and 7 were eliminated, allowed us to obtain the assignment of the coproheme propionates by resonance Raman spectroscopy and to follow the H2O2-mediated conversion of coproheme to heme b. Substitution of H2O2 by chlorite allowed us to monitor compound I formation in the inactive Y147H variant which lacks the catalytically essential Y147. This residue was demonstrated to be oxidized during turnover by using the spin-trap 2-methyl-2-nitrosopropane. Based on these findings and the data derived from molecular dynamics simulations of cofactor structures in distinct poses, we propose a reaction mechanism for the stepwise decarboxylation of coproheme that includes a 90° rotation of the intermediate three-propionate redox cofactor.

Coproheme decarboxylase (ChdC) catalyzes the oxidative decarboxylation of coproheme to heme b, i.e. the last step in the recently described coproporphyrin-dependent pathway. Coproheme decarboxylation from Listeria monocytogenes is a robust enzymatic reaction of low catalytic efficiency. Coproheme acts as both substrate and redox cofactor activated by H2O2. It fully depends on the catalytic Y147 close to the propionyl group at position 2. In the present study we have investigated the effect of disruption of the comprehensive and conserved hydrogen bonding network between the four propionates and heme cavity residues on (i) the conformational stability of the heme cavity, (ii) the electronic configuration of the ferric redox cofactor/substrate, (iii) the binding of carbon monoxide and, (iv) the decarboxylation reaction mediated by addition of H2O2. Nine single, double and triple mutants of ChdC from Listeria monocytogenes were produced in E. coli. The respective coproheme- and heme b-complexed proteins were studied by UV-Vis, resonance Raman, circular dichroism spectroscopy, and mass spectrometry. Interactions of propionates 2 and 4 with residues in the hydrophobic cavity are crucial for maintenance of the heme cavity architecture, for the mobile distal glutamine to interact with carbon monoxide, and to keep the heme cavity in a closed conformation during turnover. By contrast, the impact of substitution of residues interacting with solvent exposed propionates 6 and 7 was negligible. Except for Y147A and K151A all mutant ChdCs exhibited a wild-type-like catalytic activity. The findings are discussed with respect to the structure-function relationships of ChdCs.

Neuroglobin (Ngb) is predominantly expressed in neurons of the central and peripheral nervous systems and it clearly seems to be involved in neuroprotection. Engineering Ngb to observe structural and dynamic alterations associated with perturbation in ligand binding might reveal important structural determinants, and could shed light on key features related to its mechanism of action. Our results highlight the relevance of the CD loop and of Phe106 as distal and proximal controls involved in ligand binding in murine neuroglobin. We observed the effects of individual and combined mutations of the CD loop and Phe106 that conferred to Ngb higher CO binding velocities, which we correlate with the following structural observations: the mutant F106A shows, upon CO binding, a reduced heme sliding hindrance, with the heme present in a peculiar double conformation, whereas in the CD loop mutant “Gly-loop”, the original network of interactions between the loop and the heme was abolished, enhancing binding via facilitated gating out of the distal His64. Finally, the double mutant, combining both mutations, showed a synergistic effect on CO binding rates. Resonance Raman spectroscopy and MD simulations support our findings on structural dynamics and heme interactions in wild type and mutated Ngbs.

Significance: Temperature is one of the most important drivers in shaping protein adaptations. Many biochemical and physiological processes are influenced by temperature. Proteins and enzymes from organisms living at low temperature are less stable in comparison to high-temperature adapted proteins. The lower stability is generally due to greater conformational flexibility. Recent Advances: Adaptive changes in the structure of cold-adapted proteins may occur at subunit interfaces, distant from the active site, thus producing energy changes associated with conformational transitions transmitted to the active site by allosteric modulation, valid also for monomeric proteins in which tertiary structural changes may play an essential role. Critical Issues: Despite efforts, the current experimental and computational methods still fail to produce general principles on protein evolution, since many changes are protein and species dependent. Environmental constraints or other biological cellular signals may override the ancestral information included in the structure of the protein, thus introducing inaccuracy in estimates and predictions on the evolutionary adaptations of proteins in response to cold adaptation. Future Directions: In this review, we describe the studies and approaches used to investigate stability and flexibility in the cold-adapted globins of the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. In fact, future research directions will be prescient on more detailed investigation of cold-adapted proteins and the role of fluctuations between different conformational states. Fil: Giordano, Daniela. Institute Of Biosciences And Bioresources; Italia Fil: Boubeta, Fernando Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: di Prisco, Guido. Institute Of Biosciences And Bioresources; Italia Fil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Smulevich, Giulietta. Firenze University; Italia Fil: Viappiani, Christiano. Università di Parma; Italia Fil: Verde, Cinzia. Institute Of Biosciences And Bioresources; Italia

Structural and functional properties of ferrous Mycobacterium tuberculosis (Mt-Nb) and human (Hs-Nb) nitrobindins (Nbs) were investigated. At pH 7.0 and 25.0 °C, the unliganded Fe(II) species is penta-coordinated and unlike most other hemoproteins no pH-dependence of its coordination was detected over the pH range between 2.2 and 7.0. Further, despite a very open distal side of the heme pocket (as also indicated by the vanishingly small geminate recombination of CO for both Nbs), which exposes the heme pocket to the bulk solvent, their reactivity toward ligands, such as CO and NO, is significantly slower than in most hemoproteins, envisaging either a proximal barrier for ligand binding and/or crowding of H2O molecules in the distal side of the heme pocket which impairs ligand binding to the heme Fe-atom. On the other hand, liganded species display already at pH 7.0 and 25 °C a severe weakening (in the case of CO) and a cleavage (in the case of NO) of the proximal Fe-His bond, suggesting that the ligand-linked movement of the Fe(II) atom onto the heme plane brings about a marked lengthening of the proximal Fe-imidazole bond, eventually leading to its rupture. This structural evidence is accompanied by a marked enhancement of both ligands dissociation rate constants. As a whole, these data clearly indicate that structural–functional relationships in Nbs strongly differ from what observed in mammalian and truncated hemoproteins, suggesting that Nbs play a functional role clearly distinct from other eukaryotic and prokaryotic hemoproteins.

Glyphosate is a wide spread organophosphate herbicide whose use and associated risks are strongly debated. Many countries are regulating its use in field and it is often found in surface water. Therefore, a simple analytical method, possibly based on portable instruments, would be highly desirable. In case of many other pesticides, surface enhanced Raman scattering (SERS) spectroscopy was shown to be a very effective method to approach the problem. Glyphosate Raman and SERS spectra have been subject of different experimental and theoretical studies but still their interpretation is unclear and sensitivity in SERS experiments is quite low. In this work, SERS spectra on silver nanoparticles dispersion of glyphosate, its 2-13C isotopically substituted derivative, and its degradation product, the aminomethylphosphonic acid, have been obtained. The vibrational spectra of this latter compound allowed us to rule out a possible contribution of this species to the SERS spectra and the observed isotope shifts help to explain the SERS pattern in terms of the orientation of the adsorbed molecules on the metal surface. Therefore, we conclude that the relevant differences both on the intensities and wavenumbers between our SERS and Raman spectra of glyphosate must derive from the effect induced by the adsorption on the nanoparticles.

Coproheme decarboxylases (ChdC) catalyze the hydrogen peroxide-mediated conversion of coproheme to heme b. This work compares the structure and function of wild-type (WT) coproheme decarboxylase from Listeria monocytogenes and its M149A, Q187A, and M149A/Q187A mutants. The UV–vis, resonance Raman, and electron paramagnetic resonance spectroscopies clearly show that the ferric form of the WT protein is a pentacoordinate quantum mechanically mixed-spin state, which is very unusual in biological systems. Exchange of the Met149 residue to Ala dramatically alters the heme coordination, which becomes a 6-coordinate low spin species with the amide nitrogen atom of the Q187 residue bound to the heme iron. The interaction between M149 and propionyl 2 is found to play an important role in keeping the Q187 residue correctly positioned for closure of the distal cavity. This is confirmed by the observation that in the M149A variant two CO conformers are present corresponding to open (A0) and closed (A1) conformations. The CO of the latter species, the only conformer observed in the WT protein, is H-bonded to Q187. In the absence of the Q187 residue or in the adducts of all the heme b forms of ChdC investigated herein (containing vinyls in positions 2 and 4), only the A0 conformer has been found. Moreover, M149 is shown to be involved in the formation of a covalent bond with a vinyl substituent of heme b at excess of hydrogen peroxide.

The ligand binding characteristics of heme-containing proteins are determined by a number of factors, including the nature and conformation of the distal residues and their capability to stabilize the heme-bound ligand via hydrogen-bonding and electrostatic interactions. In this regard, the heme pockets of truncated hemoglobins (TrHbs) constitute an interesting case study as they share many common features, including a number of polar cavity residues. In this review, we will focus on three proteins of group II TrHbs, from Thermobifida fusca (Tf-HbO) and Pseudoalteromonas haloplanktis TAC125 (Ph-HbO). Although the residues in positions G8 (Trp) and B10 (Tyr) are conserved in all three proteins, the CD1 residue is a Tyr in T. fusca and a His in P. haloplanktis. Comparison of the ligand binding characteristics of these proteins, in particular the hydroxo and CO ligands by means of resonance Raman spectroscopy, reveals that this single difference in the key heme cavity residues markedly affects their ligand binding capability and conformation. Furthermore, although the two Ph-HbOs (Ph-HbO-2217 and Ph-HbO-0030) have identical key cavity residues, they display distinct ligand binding properties.

A large amount of data is currently available on the adaptive mechanisms of polar bony fish hemoglobins, but structural information on those of cartilaginous species is scarce. This study presents the first characterisation of the hemoglobin system of one of the longest-living vertebrate species (392 +/- 120 years), the Arctic shark Somniosus microcephalus. Three major hemoglobins are found in its red blood cells and are made of two copies of the same a globin combined with two copies of three very similar beta subunits. The three hemoglobins show very similar oxygenation and carbonylation properties, which are unaffected by urea, a very important compound in marine elasmobranch physiology. They display identical electronic absorption and resonance Raman spectra, indicating that their heme-pocket structures are identical or highly similar. The quaternary transition equilibrium between the relaxed (R) and the tense (T) states is more dependent on physiological allosteric effectors than in human hemoglobin, as also demonstrated in polar teleost hemoglobins. Similar to other cartilaginous fishes, we found no evidence for functional differentiation among the three isoforms. The very similar ligand-binding properties suggest that regulatory control of O-2 transport may be at the cellular level and that it may involve changes in the cellular concentrations of allosteric effectors and/or variations of other systemic factors. The hemoglobins of this polar shark have evolved adaptive decreases in O2 affinity in comparison to temperate sharks.

Despite the large number of globins recently discovered in bacteria, our knowledge of their physiological functions is restricted to only a few examples. In the microbial world, globins appear to perform multiple roles in addition to the reversible binding of oxygen; all these functions are attributable to the heme pocket that dominates functional properties. Resistance to nitrosative stress and involvement in oxygen chemistry seem to be the most prevalent functions for bacterial globins, although the number of globins for which functional roles have been studied via mutation and genetic complementation is very limited. The acquisition of structural information has considerably outpaced the physiological and molecular characterisation of these proteins. The genome of the Antarctic cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) contains genes encoding three distinct single-chain 2/2 globins, supporting the hypothesis of their crucial involvement in a number of functions, including protection against oxidative and nitrosative stress in the cold and O2-rich environment. In the genome of PhTAC125, the genes encoding 2/2 globins are constitutively transcribed, thus suggesting that these globins are not functionally redundant in their physiological function in PhTAC125. In the present study, the physiological role of one of the 2/2 globins, Ph-2/2HbO-2217, was investigated by integrating in vivo and in vitro results. This role includes the involvement in the detoxification of reactive nitrogen and O2 species including NO by developing two in vivo and in vitro models to highlight the protective role of Ph-2/2HbO-2217 against reactive nitrogen species. The PSHAa2217 gene was cloned and over-expressed in the flavohemoglobin-deficient mutant of Escherichia coli and the growth properties and O2 uptake in the presence of NO of the mutant carrying the PSHAa2217 gene were analysed. The ferric form of Ph-2/2HbO-2217 is able to catalyse peroxynitrite isomerisation in vitro, indicating its potential role in the scavenging of reactive nitrogen species. Here we present in vitro evidence for the detoxification of NO by Ph-2/2HbO-2217.

We investigate the effects of antimicrobial (sodium citrate tribasic, E331) and antioxidant (ascorbic acid, E300 and sodium ascorbate, E301) additives on the meat drip from defrosted yellowfin tuna fish loins obtained from the local market and horse heart myoglobin. The effects have been followed by electronic absorption, its second derivative spectra, and resonance Raman spectroscopies. Upon addition of the additives, a final form is reached after about 24 h. It is characterized by a 4 nm red-shifted Soret band compared to that typical of the oxy species (418 nm) but with similar Q bands. Resonance Raman experiments carried out in 16O2 and 18O2 allowed us to identify the presence of the native oxy form coexisting with a second oxygen bound species, characterized by a ν(Fe O2) stretching frequency upshifted 7 cm−1 compared to the native oxy form and with a greater (33 cm−1) isotopic shift in 18O2. These data suggest the presence of a highly bent ligand conformation. The new species induced by the addition of the additives imparts a red colour to the tuna fish meat, a characteristic that is of some concern. In fact, the presence of the new red form can mask the aging of the product that, consequently, might contain histamine. Furthermore, the electronic absorption spectrum is very similar to that of the tuna fish myoglobin carbon monoxide complex, which has important regulatory consequences. Carbon monoxide treatment of tuna is banned in the EU for masking the effects of aging on the appearance of meats.

Heme biosynthesis in Gram‐positive bacteria follows a recently described coproporphyrin‐dependent pathway with HemQ catalyzing the decarboxylation of coproheme to heme b. Here we present the first crystal structure of a HemQ (homopentameric coproheme‐HemQ from Listeria monocytogenes) at 1.69 Å resolution and the conversion of coproheme to heme b followed by UV‐vis and resonance Raman spectroscopy as well as mass spectrometry. The ferric five‐coordinated coproheme iron of HemQ is weakly bound by a neutral proximal histidine H174. In the crystal structure of the resting state, the distal Q187 (conserved in Firmicutes HemQ) is H‐bonded with propionate p2 and the hydrophobic distal cavity lacks solvent water molecules. Two H2O2 molecules are shown to be necessary for decarboxylation of the propionates p2 and p4, thereby forming the corresponding vinyl groups of heme b. The overall reaction is relatively slow (k cat/KM = 1.8 × 102 m −1·s−1 at pH 7.0) and occurs in a stepwise manner with a three‐propionate intermediate. We present the noncovalent interactions between coproheme and the protein and propose a two‐step reaction mechanism. Furthermore, the structure of coproheme‐HemQ is compared to that of the phylogenetically related heme b‐containing chlorite dismutases. Database Structural data are available in the PDB under the accession number 5LOQ.

Cytochrome c undergoes structural variations upon binding of cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several mechanisms governing cytochrome c/cardiolipin (cyt c/CL) recognition have been proposed, the interpretation of the process remains, at least in part, unknown. To better define the steps characterizing the cyt c–CL interaction, the role of Lys72 and Lys73, two residues thought to be important in the protein/lipid binding interaction, were recently investigated by mutagenesis. The substitution of the two (positively charged) Lys residues with Asn revealed that such mutations cancel the CL-dependent peroxidase activity of cyt c; furthermore, CL does not interact with the Lys72Asn mutant. In the present paper, we extend our study to the Lys→Arg mutants to investigate the influence exerted by the charge possessed by the residues located at positions 72 and 73 on the cyt c/CL interaction. On the basis of the present work a number of overall conclusions can be drawn: (i) position 72 must be occupied by a positively charged residue to assure cyt c/CL recognition; (ii) the Arg residues located at positions 72 and 73 permit cyt c to react with CL; (iii) the replacement of Lys72 with Arg weakens the second (low-affinity) binding transition; (iv) the Lys73Arg mutation strongly increases the peroxidase activity of the CL-bound protein. Graphical abstract: [Figure not available: see fulltext.]