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Ferritins are ubiquitous and conserved proteins endowed with enzymatic ferroxidase activity, that oxidize Fe(II) ions at the dimetal ferroxidase centre to form a mineralized Fe(III) oxide core deposited within the apo-protein shell. Herein, the in vitro formation of a heterodimetal cofactor constituted by Fe and Mn ions has been investigated in human H ferritin (hHFt). Namely, Mn and Fe binding at the hHFt ferroxidase centre and its effects on Fe(II) oxidation have been investigated by UV–Vis ferroxidation kinetics, fluorimetric titrations, multifrequency EPR, and preliminary Mossbauer spectroscopy. Our results show that in hHFt, both Fe(II) and Mn(II) bind the ferroxidase centre forming a Fe-Mn cofactor. Moreover, molecular oxygen seems to favour Mn(II) binding and increases the ferroxidation activity of the Mn-loaded protein. The data suggest that Mn influences the Fe binding and the efficiency of the ferroxidation reaction. The higher efficiency of the Mn-Fe heterometallic centre may have a physiological relevance in specific cell types (i.e. glia cells), where the concentration of Mn is the same order of magnitude as iron.

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 surface of gold nanorods (Au NRs) has been appropriately engineered to achieve a suitable interface for bioconjugation with horse heart cytochrome c (HCc). HCc, an extensively studied and well-characterized protein, represents an ideal model for nanoparticle (NP)–protein conjugation studies because of its small size, high stability, and commercial availability. Here, the native state of the protein has been demonstrated for the first time, by means of Raman spectroscopy, to be retained upon conjugation with the anisotropic Au nanostructures, thus validating the proposed protocol as specifically suited to mostly preserve the plasmonic properties of the NRs and to retain the structure of the protein. The successful creation of such bioconjugates with the retention of the protein structure and function along with the preservation of the NP properties represents a challenging but essential task, as it provides the only way to access functional hybrid systems with potential applications in biotechnology, medicine, and catalysis. In this perspective, the organic capping surrounding the Au NRs plays a key role, as it represents the functional interface for the conjugation step. Cetyltrimethylammonium bromide-coated Au NRs, prepared by using a seed-mediated synthetic route, have been wrapped with polyacrylic acid (PAA) by means of electrostatic interactions following a layer-by-layer approach. The resulting water-dispersible negatively charged AuNRs@PAA NPs have then been electrostatically bound to the positively charged HCc. The bioconjugation procedure has been thoroughly monitored by the combined analysis of UV–vis absorption, resonance Raman and Fourier transform infrared spectroscopies, transmission electron microscopy microscopy, and ζ-potential, which verified the successful conjugation of the protein to the nanorods.

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.

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.

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.]

Spectroscopic and functional properties of human cytochrome c and its Tyr67 residue mutants (i.e., Tyr67His and Tyr67Arg) have been investigated. In the case of the Tyr67His mutant, we have observed only a very limited structural alteration of the heme pocket and of the Ω-loop involving, among others, the residue Met80 and its bond with the heme iron. Conversely, in the Tyr67Arg mutant the Fe-Met80 bond is cleaved; consequently, a much more extensive structural alteration of the Ω-loop can be envisaged. The structural, and thus the functional modifications, of the Tyr67Arg mutant are present in both the ferric [Fe(III)] and the ferrous [Fe(II)] forms, indicating that the structural changes are independent of the heme iron oxidation state, depending instead on the type of substituting residue. Furthermore, a significant peroxidase activity is evident for the Tyr67Arg mutant, highlighting the role of Arg as a basic, positively charged residue at pH7.0, located in the heme distal pocket, which may act as an acid to cleave the O-O bond in H2O2. As a whole, our results indicate that a delicate equilibrium is associated with the spatial arrangement of the Ω-loop. Clearly, Arg, but not His, is able to stabilize and polarize the negative charge on the Fe(III)-OOH complex during the formation of Compound I, with important consequences on cytochrome peroxidation activity and its role in the apoptotic process, which is somewhat different in yeast and mammals.

Fep1, the iron-responsive GATA factor from the methylotrophic yeast Pichia pastoris, has been characterised both in vivo and in vitro. This protein has two Cys2-Cys2 type zinc fingers and a set of four conserved cysteines arranged in a Cys-X5-Cys-X8-Cys-X2-Cys motif located between the two zinc fingers. Electronic absorption and resonance Raman spectroscopic analyses in anaerobic and aerobic conditions indicate that Fep1 binds iron in the form of a [2Fe-2S] cluster. Site-directed mutagenesis shows that replacement of the four cysteines with serine inactivates this transcriptional repressor. Unexpectedly, the inactive mutant is still able to bind a [2Fe-2S] cluster, employing two cysteine residues belonging to the first zinc finger. These two cysteine residues can act as alternative cluster ligands selectively in aerobically purified Fep1 wild type, suggesting that oxygen could play a role in Fep1 function by causing differential localization of the [Fe-S] cluster.

The blood of the sub-Antarctic fish Eleginops maclovinus (Em) contains three haemoglobins. The major haemoglobin (Hb1Em) displays the Root effect, a drastic decrease in the oxygen affinity and a loss of cooperativity at acidic pH. The carbomonoxy form of HbEm1 has been crystallized in two different crystal forms, orthorhombic (Ortho) and hexagonal (Hexa), and high-resolution diffraction data have been collected for both forms (1.45 and 1.49 A ˚ resolution, respectively). The high-frequency resonance Raman spectra collected from the two crystal forms using excitation at 514 nm were almost indistinguishable. Hb1Em is the first sub-Antarctic fish Hb to be crystallized and its structural characterization will shed light on the molecular mechanisms of cold adaptation and the role of the Root effect in fish haemoglobins.

Dehaloperoxidase (DHP) from the annelid Amphitrite ornata is a catalytically active hemoglobin-peroxidase that possesses a unique internal binding cavity in the distal pocket above the heme. The previously published crystal structure of DHP shows 4-iodophenol bound internally. This led to the proposal that the internal binding site is the active site for phenol oxidation. However, the native substrate for DHP is 2,4,6-tribromophenol, and all attempts to bind 2,4,6-tribromophenol in the internal site under physiological conditions have failed. Herein, we show that the binding of 4-halophenols in the internal pocket inhibits enzymatic function. Furthermore, we demonstrate that DHP has a unique two-site competitive binding mechanism in which the internal and external binding sites communicate through two conformations of the distal histidine of the enzyme, resulting in nonclassical competitive inhibition. The same distal histidine conformations involved in DHP function regulate oxygen binding and release during transport and storage by hemoglobins and myoglobins. This work provides further support for the hypothesis that DHP possesses an external binding site for substrate oxidation, as is typical for the peroxidase family of enzymes.

The present work highlights the important role played by the distal histidine in controlling the binding of heme ligands in dehaloperoxidase (DHP) as compared to myoglobin and peroxidases. In DHP the distal histidine is highly mobile and undergoes a conformational change that places it within hydrogen-bonding distance of anionic ligands and water, where strong hydrogen bonding can occur. The detailed resonance Raman (RR) analysis at room temperature shows the presence of an equilibrium between a 5-coordinate and a 6-coordinate (aquo) high-spin form. The equilibrium shifts toward the aquo form at 12 K. These two forms are consistent with the existing X-ray structures where a closed conformation, with His55 positioned in the distal pocket and H-bonded with the distal water molecule (6-coordinate), and an open solvent-exposed conformation, with the His55 displaced from the distal pocket (5-coordinate form), are in equilibrium. Moreover, the comparison between the Raman data at 298 and 12 K and the results obtained by EPR of DHP in the presence of 4-iodophenol highlights the formation of a pure 5-coordinate high-spin form (open conformation). The data reported herein support the role of His55 in facilitating the interaction of substrate and inhibitor in the regulation of enzyme function, as previously suggested. The two conformations of His55 in equilibrium at room temperature provide a level of control that permits the distal histidine to act as both the acid-base catalyst in the peroxidase mechanism and the stabilizing amino acid for exogenous heme-coordinated ligands.

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.

The heme-containing peroxidases of the plant peroxidase superfamily have been the subject of extensive studies to gain a complete understanding of their structure and function properties. Spectroscopic techniques have been fundamental to the comprehension of peroxidase function under physiological conditions. Resonance Raman spectroscopy has assumed a particularly important role due to its extreme sensitivity to alterations in the surroundings of the heme, which makes it an ideal tool for studying molecular interactions at the peroxidase active sites. In combination with site-directed mutagenesis and X-ray structural data, it has given insight into the influence exerted on protein architecture in the vicinity of the active site by key amino acids and furnished critical information on subtle structural features important for protein flexibility, functionality and stability. The mutation of key catalytic residues on both the distal and proximal sides of the heme cavity not only revealed their roles in the reaction mechanism of peroxidases, but also demonstrated that in peroxidases there are common structural mechanisms which facilitate communication between the two sides of the heme cavity and highlighted the importance of long range interactions in maintaining the functional properties of the heme. The overwhelming success of this approach using the combination of site-directed mutagenesis together with spectroscopic techniques and X-ray structural data to unveil detailed aspects of the structure–function relationships of heme peroxidases, firstly in the pioneering studies on yeast cytochrome c peroxidase then for other peroxidases, has led to its subsequent widespread general application in the characterization of heme proteins.

Truncated hemoglobins build one of the three branches of the globin protein superfamily. They display a characteristic two‐on‐two α‐helical sandwich fold and are clustered into three groups (I, II and III) based on distinct structural features. Truncated hemoglobins are present in eubacteria, cyanobacteria, protozoa and plants. Here we present a structural, spectroscopic and molecular dynamics characterization of a group‐II truncated hemoglobin, encoded by the PSHAa0030 gene from Pseudoalteromonas haloplanktis TAC125 (Ph‐2/2HbO), a cold‐adapted Antarctic marine bacterium hosting one flavohemoglobin and three distinct truncated hemoglobins. The Ph‐2/2HbO aquo‐met crystal structure (at 2.21 Å resolution) shows typical features of group‐II truncated hemoglobins, namely the two‐on‐two α‐helical sandwich fold, a helix Φ preceding the proximal helix F, and a heme distal‐site hydrogen‐bonded network that includes water molecules and several distal‐site residues, including His(58)CD1. Analysis of Ph‐2/2HbO by electron paramagnetic resonance, resonance Raman and electronic absorption spectra, under varied solution conditions, shows that Ph‐2/2HbO can access diverse heme ligation states. Among these, detection of a low‐spin heme hexa‐coordinated species suggests that residue Tyr(42)B10 can undergo large conformational changes in order to act as the sixth heme‐Fe ligand. Altogether, the results show that Ph‐2/2HbO maintains the general structural features of group‐II truncated hemoglobins but displays enhanced conformational flexibility in the proximity of the heme cavity, a property probably related to the functional challenges, such as low temperature, high O2 concentration and low kinetic energy of molecules, experienced by organisms living in the Antarctic environment. Fil: Giordano, Daniela. Consiglio Nazionale delle Ricerche; Italia Fil: Pesce, Alessandra. Università degli Studi di Genova; Italia Fil: Boechi, Leonardo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bustamante, Juan Pablo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Caldelli, Elena. Universita Degli Studi Di Firenze; Italia Fil: Howes, Barry D.. Universita Degli Studi Di Firenze; Italia Fil: Riccio, Alessia. Consiglio Nazionale delle Ricerche; Italia Fil: di Prisco, Guido. Consiglio Nazionale delle Ricerche; Italia Fil: Nardini, Marco. Università degli Studi di Milano; Italia Fil: Estrin, Dario Ariel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Smulevich, Giulietta. Universita Degli Studi Di Firenze; Italia Fil: Bolognesi, Martino. Università degli Studi di Milano; Italia Fil: Verde, Cinzia. Consiglio Nazionale delle Ricerche; Italia. Universita Di Roma; Italia

Protein-bound iron sulfur clusters are prosthetic groups involved in several metabolic pathways. Understanding how they interact with the host protein and which factors influence their stability is therefore an important goal in biology. Here, we have addressed this question by studying the determinants of the 2Fe–2S cluster stability in the IscU/Isu protein scaffold. Through a detailed computational study based on a mixed quantum and classical mechanics approach, we predict that the simultaneous presence of two conserved residues, D39 and H105, has a conflicting role in cluster coordination which results in destabilizing cluster-loaded IscU/Isu according to a ‘tug-of-war’ mechanism. The effect is absent in the D39A mutant already known to host the cluster more stably. Our theoretical conclusions are directly supported by experimental data, also obtained from the H105A mutant, which has properties intermediate between the wild-type and the D39A mutant. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

In this chapter, we will discuss the paradigmatic case of Thermobifida fusca (Tf-trHb) HbO in its ferrous and ferric states and its behaviour towards a battery of possible ligands. This choice was dictated by the fact that it has been one of the most extensively studied truncated haemoglobins, both in terms of spectroscopic and molecular dynamics studies. Tf-trHb typifies the structural properties of group II trHbs, as the active site is characterized by a highly polar distal environment in which TrpG8, TyrCD1, and TyrB10 provide three potential H-bond donors in the distal cavity capable of stabilizing the incoming ligands. The role of these residues in key topological positions, and their interplay with the iron-bound ligands, has been addressed in studies carried out on the CO, F(-), OH(-), CN(-), and HS(-) adducts formed with the wild-type protein and a combinatorial set of mutants, in which the distal polar residues, TrpG8, TyrCD1, and TyrB10, have been singly, doubly, or triply replaced by a Phe residue. In this context, such a complete analysis provides an excellent benchmark for the investigation of the relationship between protein structure and function, allowing one to translate physicochemical properties of the active site into the observed functional behaviour. Tf-trHb will be compared with other members of the group II trHbs and, more generally, with members of the other trHb subgroups.

The unique architecture of the active site of Thermobifida fusca truncated hemoglobin (Tf-trHb) and other globins belonging to the same family has stimulated extensive studies aimed at understanding the interplay between iron-bound ligands and distal amino acids. The behavior of the heme-bound hydroxyl, in particular, has generated much interest in view of the relationships between the spin-state equilibrium of the ferric iron atom and hydrogen-bonding capabilities (as either acceptor or donor) of the OH− group itself. The present investigation offers a detailed molecular dynamics and spectroscopic picture of the hydroxyl complexes of the WT protein and a combinatorial set of mutants, in which the distal polar residues, TrpG8, TyrCD1, and TyrB10, have been singly, doubly, or triply replaced by a Phe residue. Each mutant is characterized by a complex interplay of interactions in which the hydroxyl ligand may act both as a H-bond donor or acceptor. The resonance Raman stretching frequencies of the Fe−OH moiety, together with electron paramagnetic resonance spectra and MD simulations on each mutant, have enabled the identification of specific contributions to the unique ligand-inclusive H-bond network typical of this globin family. Fil: Nicoletti, Francesco P.. Universita Degli Studi Di Firenze; Italia Fil: Bustamante, Juan Pablo. 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: Droghetti, Enrica. Universita Degli Studi Di Firenze; Italia Fil: Howes, Barry D.. Universita Degli Studi Di Firenze; Italia Fil: Fittipaldi, Maria. Universita Degli Studi Di Firenze; Italia Fil: Bonamore, Alessandra. Universita Di Roma; Italia Fil: Baiocco, Paola. Italian Institute of Technology; Italia Fil: Feis, Alessandro. Universita Degli Studi Di Firenze; Italia Fil: Boffi, Alberto. Universita Di Roma; 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. Universita Degli Studi Di Firenze; Italia

Studies on horseradish peroxidase C and other haem peroxidases have been carried out on selected mutants in the distal haem cavity providing insight into the functional importance of the distal residues. Recent work has demonstrated that proximal structural features can also exert an important influence in determining the electronic structure of the haem pocket. To extend our understanding of the significance of proximal characteristics in regulating haem properties the proximal Thr171Ser mutant has been constructed. Thr171 is an important linking residue between the structural proximal Ca2+ ion and the proximal haem ligand, in particular the methyl group of Thr171 interdigitates with other proximal residues in the core of the enzyme. Although the mutation induces no significant changes to the functional properties of the enzyme, electronic absorption and resonance Raman spectroscopy reveal that it has a highly selective affect on the reduced state of the enzyme, effectively stabilizing it, whilst the electronic properties of the Fe(III) state unchanged and essentially identical to those of the native protein. This results in a significant change in the Fe2+/Fe3+ redox potential of the mutant. It is concluded that the unusual properties of the Thr171Ser mutant reflect the loss of a structural restraint in the proximal haem pocket that allows 'slippage' of the proximal haem ligand, but only in the reduced state. This is a remarkably subtle and specific effect that appears to increase the flexibility of the reduced state of the mutant compared to that of the wild-type protein.

The discovery that cysteine (Cys) S-nitrosation of trout myoglobin (Mb) increases heme O2 affinity has revealed a novel allosteric effect that may promote hypoxia-induced nitric oxide (NO) delivery in the trout heart and improve myocardial efficiency. To better understand this allosteric effect, we investigated the functional effects and structural origin of S-nitrosation in selected fish Mbs differing by content and position of reactive cysteine (Cys) residues. The Mbs from the Atlantic salmon and the yellowfin tuna, containing two and one reactive Cys, respectively, were S-nitrosated in vitro by reaction with Cys-NO to generate Mb-SNO to a similar yield (∼0.50 SH/heme), suggesting reaction at a specific Cys residue. As found for trout, salmon Mb showed a low O2 affinity (P 50 = 2.7 torr) that was increased by S-nitrosation (P 50 = 1.7 torr), whereas in tuna Mb, O2 affinity (P 50 = 0.9 torr) was independent of S-nitrosation. O2 dissociation rates (k off) of trout and salmon Mbs were not altered when Cys were in the SNO or N-ethylmaleimide (NEM) forms, suggesting that S-nitrosation should affect O2 affinity by raising the O2 association rate (k on). Taken together, these results indicate that O2-linked S-nitrosation may occur specifically at Cys107, present in salmon and trout Mb but not in tuna Mb, and that it may relieve protein constraints that limit O2 entry to the heme pocket of the unmodified Mb by a yet unknown mechanism. UV-Vis and resonance Raman spectra of the NEM-derivative of trout Mb (functionally equivalent to Mb-SNO and not photolabile) were identical to those of the unmodified Mb, indicating that S-nitrosation does not affect the extent or nature of heme-ligand stabilization of the fully ligated protein. The importance of S-nitrosation of Mb in vivo is confirmed by the observation that Mb-SNO is present in trout hearts and that its level can be significantly reduced by anoxic conditions.

The extent to which the structural Ca(2+) ions of horseradish peroxidase (HRPC) are a determinant in defining the heme pocket architecture is investigated by electronic absorption and resonance Raman spectroscopy upon removal of one Ca(2+) ion. The Fe(III) heme states are modified upon Ca(2+) depletion, with an uncommon quantum mechanically mixed spin state becoming the dominant species. Ca(2+)-depleted HRPC forms complexes with benzohydroxamic acid and CO which display spectra very similar to those of native HRPC, indicating that any changes to the distal cavity structural properties upon Ca(2+) depletion are easily reversed. Contrary to the native protein, the Ca(2+)-depleted ferrous form displays a low-spin bis-histidyl heme state and a small proportion of high-spin heme. Furthermore, the nu(Fe-Im) stretching mode downshifts 27 cm(-1) upon Ca(2+) depletion revealing a significant structural perturbation of the proximal cavity near the histidine ligand. The specific activity of the Ca(2+)-depleted enzyme is 50% that of the native form. The effects on enzyme activity and spectral features observed upon Ca(2+) depletion are reversible upon reconstitution. Evaluation of the present and previous data firmly favors the proximal Ca(2+) ion as that which is lost upon Ca(2+) depletion and which likely plays the more critical role in regulating the heme pocket structural and catalytic properties.

Aromatic substrate binding to peroxidases is mediated through hydrophobic and hydrogen bonding interactions between residues on the distal side of the heme and the substrate molecule. The effects of perturbing these interactions are investigated by an electronic absorption and resonance Raman study of benzohydroxamic acid (BHA) binding to a series of mutants of horseradish peroxidase isoenzyme C (HRPC). In particular, the Phe179 Ala, His42 Glu variants and the double mutant His42 Glu:Arg38 Leu are studied in their ferric state at pH 7 with and without BHA. A comparison of the data with those previously reported for wild-type HRPC and other distal site mutants reaffirms that in the resting state mutation of His42 leads to an increase of 6-coordinate aquo heme forms at the expense of the 5-coordinate heme state, which is the dominant species in wild-type HRPC. The His42Glu:Arg38Leu double mutant displays an enhanced proportion of the pentacoordinate heme state, similar to the single Arg38Leu mutant. The heme spin states are insensitive to mutation of the Phe179 residue. The BHA complexes of all mutants are found to have a greater amount of unbound form compared to the wild-type HRPC complex. It is apparent from the spectral changes induced on complexation with BHA that, although Phe179 provides an important hydrophobic interaction with BHA, the hydrogen bonds formed between His42 and, in particular, Arg38 and BHA assume a more critical role in the binding of BHA to the resting state. © 2001 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 62: 261–267, 2001

A distinctive characteristic of the class III heme peroxidases of the plant peroxidase superfamily is the presence of a pentacoordinate quantum mechanically mixed-spin heme state resulting from the admixture of S = 5/2 and S = 3/2 states. This is not observed in class I or II peroxidases and, in fact, is a very rare heme spin state. The corresponding hexacoordinate quantum mechanically mixed-spin state is even more uncommon and has not been observed in heme proteins. The presence of the pentacoordinate form in the class III peroxidases suggested that they could be ideal candidates to display also the six-coordinate quantum mechanically mixed-spin state. With this possibility in mind, the benzohydroxamic acid complexes of the class III peroxidases horseradish isoenzyme C and A2 and soybean peroxidase are studied by electronic absorption, resonance Raman, and EPR spectroscopy at room and low temperatures. The results are compared with those obtained for Coprinus cinereus peroxidase which belongs to class II...

Electronic absorption and resonance Raman (RR) spectra of the ferric form of barley grain peroxidase (BP 1) at various pH values, at both room temperature and 20K, are reported, together with electron paramagnetic resonance spectra at 10K. The ferrous forms and the ferric complex with fluoride have also been studied. A quantum mechanically mixed-spin (QS) state has been identified. The QS heme species coexists with 6- and 5-cHS hemes; the relative populations of these three spin states are found to be dependent on pH and temperature. However, the QS species remains in all cases the dominant heme spin species. Barley peroxidase appears to be further characterized by a splitting of the two vinyl stretching modes, indicating that the vinyl groups are differently conjugated with the porphyrin. An analysis of the currently available spectroscopic data for proteins from all three peroxidase classes suggests that the simultaneous occurrence of the QS heme state as well as the splitting of the two vinyl stretching modes is confined to class III enzymes. The former point is discussed in terms of the possible influences of heme deformations on heme spin state. It is found that moderate saddling alone is probably not enough to cause the QS state, although some saddling may be necessary for the QS state.

The resonance Raman (RR) and electronic absorption spectra of ferric horseradish peroxidase isozyme A2 (HRPA2) and of its complex with fluoride are reported. The data show the presence of a five-coordinate quantum-mixed spin (QS) species, resulting from the admixture of intermediate-spin, S=3/2, and high-spin, S=5/2, configurations, coexisting with a five- and a six-coordinate high-spin species. Fluoride binds almost completely, giving rise to a six-coordinate high-spin heme. In the RR spectra of both the resting enzyme and fluoride complex two ν(C=C) stretching modes of the vinyl substituents are observed at 1622 and 1631 cm-1. A comparison of the data obtained for HRPA2 with the electronic and RR spectra of soybean and barley peroxidases, all belonging to class III of the ‘superfamily of plant peroxidases,’ shows that the QS species is common to all these proteins. Furthermore, it has allowed us to infer that the QS heme is characterized by very high wavenumbers of the core-size marker bands and an electronic absorption spectrum blue-shifted with respect to those of the high-spin hemes. © 1998 John Wiley & Sons, Ltd.

The manner in which the distal heme pocket residues of peroxidases control the reaction mechanism and ligand binding has been investigated further by analysis of the electronic absorption and resonance Raman (RR) spectra of distal site mutants of recombinant horseradish peroxidase (HRP-C*). The roles of the conserved distal histidine and arginine residues, particularly in the context of the catalytic mechanism originally proposed for cytochrome c peroxidase (CCP), have been evaluated by studying the His42 --Leu, His42 --Arg, Arg38 --Gly, and Arg38 --Leu variants of HRP-C*. Spectra of the ferric forms, their complexes with benzohydroxamic acid (BHA), and the ferrous forms have been recorded at neutral pH. In addition, the ferric forms have been studied at alkaline pH. The relative populations of the three heme spin states characteristic of HRP-C* and its mutants were found to vary markedly from mutant to mutant. This diversity of heme spin state populations among the various mutants has allowed a well-defined set of RR frequencies to be compiled for the three heme spin states. These frequencies support the analysis of wild-type HRP-C* in terms of two heme states, five- (5cHS#) and six-coordinate high-spin (6cHS#), which exhibit anomalous RR frequencies compared to those of model heme systems. The third heme spin state is identified as being six-coordinate high-spin, displaying typical RR frequencies (6cHS). The 6cHS# and the 6cHS heme states are characterized by H bonding between the iron-bound water molecule and the Arg38 residue or the His42 residue, respectively. The proportion of six-coordinate high-spin heme states is at a minimum in the Arg38Leu mutant, indicating that the occupancy of the distal water molecule site is reduced in this mutant. The His42Arg mutant is distinguished from the other mutants by the unexpected presence of an iron-bound hydroxyl group at neutral pH. The spectral changes induced upon complexation with BHA indicate that both the distal histidine and arginine are involved in BHA binding; however, the arginine residue appears to play a more critical role. Measurements at pH 12 suggest there is a concerted involvement of both distal residues in mediating the alkaline transition of HRP-C*. Arg38 appears to be essential for stabilization of the OH- ligand, while His42 acts as a H bond acceptor. A striking similarity between the roles of these residues in the reaction of H2O2 with the enzyme and the alkaline transition is noted. By comparison with the results from corresponding mutants of CCP, it appears that although the hydrogen-bonding network linking the distal and proximal sides of the heme is conserved the distal cavity in HRP-C differs significantly from that of CCP. However, some similarities in the local environment of the distal arginine are suggested.

The ferric form of truncated hemoglobin II from Thermobifida fusca (Tf-trHb) and its triple mutant WG8F-YB10F-YCD1F at neutral and alkaline pH, and in the presence of CN− have been characterized by resonance Raman spectroscopy, electron paramagnetic resonance spectroscopy, and molecular dynamics simulations. Tf-trHb contains three polar residues in the distal site, namely TrpG8, TyrCD1 and TyrB10. Whereas TrpG8 can act as a potential hydrogen-bond donor, the tyrosines can act as donors or acceptors. Ligand binding in heme-containing proteins is 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. Since both the RR Fe-OH− and Fe-CN− frequencies are very sensitive to the distal environment, detailed information on structural variations has been obtained. The hydroxyl ligand binds only the WT protein giving rise to two different conformers. In form 1 the anion is stabilized by H-bonds with TrpG8, TyrCD1 and a water molecule, in turn H-bonded to TyrB10. In form 2, H-bonding with TyrCD1 is mediated by a water molecule. Unlike the OH− ligand, CN− binds both WT and the triple mutant giving rise to two forms with similar spectroscopic characteristics. The overall results clearly indicate that H-bonding interactions both with distal residues and water molecules are important structural determinants in the active site of Tf-trHb. Fil: Nicoletti, Francesco P.. Universita Degli Studi Di Firenze; Italia Fil: Droghetti, Enrica. Universita Degli Studi Di Firenze; Italia Fil: Howes, Barry D.. Universita Degli Studi Di Firenze; Italia Fil: Bustamante, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de Los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires; Argentina Fil: Bonamore, Alessandra. Universita Di Roma; Italia Fil: Sciamanna, Natascia. Universita Di Roma; 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; Argentina. Universidad de Buenos Aires; Argentina Fil: Feis, Alessandro. Universita Degli Studi Di Firenze; Italia Fil: Boffi, Alberto. Universita Di Roma; Italia. Centro Nazionale di Ricerca. Institute Pasteur; Italia Fil: Smulevich, Giulietta. Universita Degli Studi Di Firenze; Italia

Cytochrome c undergoes structural variations during the apoptotic process; such changes have been related to modifications occurring in the protein when it forms a complex with cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several studies have been performed to identify the site(s) of the protein involved in the cytochrome c-cardiolipin interaction, to date the location of this hosting region(s) remains unidentified and is a matter of debate. To gain deeper insight into the reaction mechanism, we investigate the role that the Lys72, Lys73, and Lys79 residues play in the cytochrome c-cardiolipin interaction, as these side chains appear to be critical for cytochrome c-cardiolipin recognition. The Lys72Asn, Lys73Asn, Lys79Asn, Lys72/73Asn, and Lys72/73/79Asn mutants of horse heart cytochrome c were produced and characterized by circular dichroism, ultraviolet-visible, and resonance Raman spectroscopies, and the effects of the mutations on the interaction of the variants with cardiolipin have been investigated. The mutants are characterized by a subpopulation with non-native axial coordination and are less stable than the wild-type protein. Furthermore, the mutants lacking Lys72 and/or Lys79 do not bind cardiolipin, and those lacking Lys73, although they form a complex with the phospholipid, do not show any peroxidase activity. These observations indicate that the Lys72, Lys73, and Lys79 residues stabilize the native axial Met80-Fe(III) coordination as well as the tertiary structure of cytochrome c. Moreover, while Lys72 and Lys79 are critical for cytochrome c-cardiolipin recognition, the simultaneous presence of Lys72, Lys73, and Lys79 is necessary for the peroxidase activity of cardiolipin-bound cytochrome c.

The genome of the cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 contains multiple genes encoding three distinct monomeric hemoglobins exhibiting a 2/2 α-helical fold. In the present work, one of these hemoglobins is studied by resonance Raman, electronic absorption and electronic paramagnetic resonance spectroscopies, kinetic measurements, and different bioinformatic approaches. It is the first cold-adapted bacterial hemoglobin to be characterized. The results indicate that this protein belongs to the 2/2 hemoglobin family, Group II, characterized by the presence of a tryptophanyl residue on the bottom of the heme distal pocket in position G8 and two tyrosyl residues (TyrCD1 and TyrB10). However, unlike other bacterial hemoglobins, the ferric state, in addition to the aquo hexacoordinated high-spin form, shows multiple hexacoordinated low-spin forms, where either TyrCD1 or TyrB10 can likely coordinate the iron. This is the first example in which both TyrCD1 and TyrB10 are proposed to be the residues that are alternatively involved in heme hexacoordination by endogenous ligands.

International audience; Progress in understanding the mechanism underlying the enzymatic formation of iron-sulfur clusters is difficult since it involves a complex reaction and a multi-component system. By exploiting different spectroscopies, we characterize the effect on the enzymatic kinetics of cluster formation of CyaY, the bacterial ortholog of frataxin, on cluster formation on the scaffold protein IscU. Frataxin/CyaY is a highly conserved protein implicated in an incurable ataxia in humans. Previous studies had suggested a role of CyaY as an inhibitor of iron sulfur cluster formation. Similar studies on the eukaryotic proteins have however suggested for frataxin a role as an activator. Our studies independently confirm that CyaY slows down the reaction and shed new light onto the mechanism by which CyaY works. We observe that the presence of CyaY does not alter the relative ratio between [2Fe2S](2+) and [4Fe4S](2+) but directly affects enzymatic activity.

Two models have been proposed to explain the interaction of cytochrome c with cardiolipin (CL) vesicles. In one case, an acyl chain of the phospholipid accommodates into a hydrophobic channel of the protein located close the Asn52 residue, whereas the alternative model considers the insertion of the acyl chain in the region of the Met80-containing loop. In an attempt to clarify which proposal offers a more appropriate explanation of cytochrome c-CL binding, we have undertaken a spectroscopic and kinetic study of the wild type and the Asn52Ile mutant of iso-1-cytochrome c from yeast to investigate the interaction of cytochrome c with CL vesicles, considered here a model for the CL-containing mitochondrial membrane. Replacement of Asn52, an invariant residue located in a small helix segment of the protein, may provide data useful to gain novel information on which region of cytochrome c is involved in the binding reaction with CL vesicles. In agreement with our recent results revealing that two distinct transitions take place in the cytochrome c-CL binding reaction, data obtained here support a model in which two (instead of one, as considered so far) adjacent acyl chains of the liposome are inserted, one at each of the hydrophobic sites, into the same cytochrome c molecule to form the cytochrome c-CL complex.

Recent studies have demonstrated that hemoglobins isolated from Antarctic fish undergo peculiar oxidation processes. Here we show, by combining crystallographic and spectroscopic data, that the oxidation pathway of Trematomus bernacchii hemoglobin (HbTb) is distinct from that observed for the major component of Trematomus newnesi (Hb1Tn), despite the high sequence identity of the two proteins and structural similarity of their ferrous and fully oxidized states. Resonance Raman analysis of HbTb autoxidation upon air-exposure reveals the absence of the oxidized pentacoordinated state that was observed for Hb1Tn. The HbTb oxidation pathway is characterized by two ferric species: an aquo hexacoordinated high spin state and a bis-histidyl hexacoordinated low spin form, which appear in the early stages of the oxidation process. The high resolution structure of an intermediate along the oxidation pathway has been determined at 1.4 A resolution. The analysis of the electron density of the heme pocket shows, for both the α and the β iron, the coexistence of multiple binding states. In this partially oxidized form, HbTb exhibits significant deviations from the canonical R state both at the local and global level. The analysis of these modifications highlights the structural correlation between key functional regions of the protein. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 1117–1125, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

31P ENDOR spectra are described for three different molybdenum(V) species in reduced xanthine oxidase samples. The spectra were not affected by removing the FAD from the enzyme, implying that this is located at some distance from molybdenum. Furthermore, in confirmation of the work of J. L. Johnson, R. E. London, and K. V. Rajagopalan [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6493-6497], NMR and chemical analysis of the phosphate content of highly purified xanthine oxidase showed there are only three phosphate residues per subunit of the enzyme. It is concluded that the ENDOR features are due to hyperfine coupling of the phosphate group of the pterin cofactor to the molybdenum atom. Evaluation of the dipolar component of the coupling has permitted estimation of the molybdenum-phosphorus distances as 7-12 A. This implies that the cofactor is in an extended conformation in the enzyme molecule. Less detailed 31P ENDOR data on sulfite oxidase are consistent with a similar conformation for the cofactor in this enzyme.

The photosystem II subunit PsbS is essential for excess energy dissipation (qE); however, both lutein and zeaxanthin are needed for its full activation. Based on previous work, two models can be proposed in which PsbS is either 1) the gene product where the quenching activity is located or 2) a proton-sensing trigger that activates the quencher molecules. The first hypothesis requires xanthophyll binding to two PsbS-binding sites, each activated by the protonation of a dicyclohexylcarbodiimide-binding lumen-exposed glutamic acid residue. To assess the existence and properties of these xanthophyll-binding sites, PsbS point mutants on each of the two Glu residues PsbS E122Q and PsbS E226Q were crossed with the npq1/npq4 and lut2/npq4 mutants lacking zeaxanthin and lutein, respectively. Double mutants E122Q/npq1 and E226Q/npq1 had no qE, whereas E122Q/lut2 and E226Q/lut2 showed a strong qE reduction with respect to both lut2 and single glutamate mutants. These findings exclude a specific interaction between lutein or zeaxanthin and a dicyclohexylcarbodiimide-binding site and suggest that the dependence of nonphotochemical quenching on xanthophyll composition is not due to pigment binding to PsbS. To verify, in vitro, the capacity of xanthophylls to bind PsbS, we have produced recombinant PsbS refolded with purified pigments and shown that Raman signals, previously attributed to PsbS-zeaxanthin interactions, are in fact due to xanthophyll aggregation. We conclude that the xanthophyll dependence of qE is not due to PsbS but to other pigment-binding proteins, probably of the Lhcb type.

Non-symbiotic hemoglobins are hexacoordinated heme proteins found in all plants. To gain insight into the importance of the heme hexacoordination and the coordinated distal histidine in general for the possible physiological functions of these proteins, the distal His(E7) of Arabidopsis thaliana hemoglobin (AHb1) was substituted by a leucine residue. The heme properties of the wild-type and mutant proteins have been characterized by electronic absorption, resonance Raman and electron paramagnetic resonance spectroscopic studies at room and low temperatures. Significant differences between the wild-type and mutant proteins have been detected. The most striking is the formation of an uncommon quantum mechanically mixed-spin heme species in the mutant. This is the first observation of such a spin state in a plant hemoglobin. The proportion of this species, which at room temperature coexists with a minor pentacoordinated high-spin form, increases markedly at low temperature.

In this paper we investigate the role played by each histidine in the amino acid sequence of yeast iso-1-cytochrome c (with the exception of H18, the residue axially coordinated to the heme iron) in determining the protein structure and stability. To this end, we have generated and characterized the double mutants H26Y/H33Y, H26Y/H39K and H33Y/H39K obtained from the C102T variant of the protein, which retain only one histidine side chain in the amino acid sequence. In particular, the H39K mutation inserts a lysine at position 39 as in the sequence of equine cytochrome c. The H26Y/H33Y/H39K triple mutant, which lacks all three histidines, was also produced and its spectroscopic properties are compared with those of the double mutants. The data highlight the critical role played by H26 in determining protein stability. Recombinant horse cytochrome c and the corresponding H26Y mutant were also generated and characterized. Since equine cytochrome c exhibits higher stability than the yeast protein, this provides a valuable opportunity to understand the role played by the invariant H26 residue in determining structure and stability.

Spectroscopic techniques have been fundamental to the comprehension of peroxidase function under physiological conditions. This Account examines the contribution to our understanding of heme peroxidases provided by electronic and resonance Raman spectroscopies in conjunction with site-directed mutagenesis. The results obtained over 15 years with several heme peroxidases and selected mutants have provided important insights into the influence exerted by the protein in the vicinity of the active site via key amino acids on the functionality and stability of the enzymes. Moreover, resonance Raman spectroscopy has revealed that a common feature of heme peroxidases is the presence of an extensive network of H-bonds coupling the distal and proximal sides, which has a profound influence on the heme ligation, affecting both the fifth and the sixth coordination sites.

The suborder Notothenioidei dominates the Antarctic ichthyofauna. The non-Antarctic monotypic family Pseudaphritidae is one of the most primitive families. The characterization of the oxygen-transport system of euryhaline Pseudaphritis urvillii is herewith reported. Similar to most Antarctic notothenioids, this temperate species has a single major hemoglobin (Hb 1, over 95% of the total). Hb 1 has strong Bohr and Root effects. It shows two very uncommon features in oxygen binding: At high pH values, the oxygen affinity is exceptionally high compared to other notothenioids, and subunit cooperativity is modulated by pH in an unusual way, namely the curve of the Hill coefficient is bell-shaped, with values approaching 1 at both extremes of pH. Molecular modeling, electronic absorption and resonance Raman spectra have been used to characterize the heme environment of Hb 1 in an attempt to explain these features, particularly in view of some potentially important nonconservative replacements found in the primary structure. Compared to human HbA, no major changes were found in the structure of the proximal cavity of the alpha-chain of Hb 1, although an altered distal histidyl and heme position was identified in the models of the beta-chain, possibly facilitated by a more open heme pocket due to reduced steric constraints on the vinyl substituent groups. This conformation may lead to the hemichrome form identified by spectroscopy in the Met state, which likely fulfils a potentially important physiological role.

The structural and redox properties of a non-covalent complex reconstituted upon mixing two non-contiguous fragments of horse cytochrome c, the residues 1-38 heme-containing N-fragment with the residues 57-104 C-fragment, have been investigated. With respect to native cyt c, the complex lacks a segment of 18 residues, corresponding, in the native protein, to an omega (Omega)-loop region. The fragment complex shows compact structure, native-like alpha-helix content but a less rigid atomic packing and reduced stability with respect to the native protein. Structural heterogeneity is observed at pH 7.0, involving formation of an axially misligated low-spin species and consequent partial displacement of Met80 from the sixth coordination position of the heme-iron. Spectroscopic data suggest that a lysine (located in the Met80-containing loop, namely Lys72, Lys73, or Lys79) replaces the methionine residue. The residues 1-38/57-104 fragment complex shows an unusual biphasic alkaline titration characterized by a low (p K(a1)=6.72) and a high p K(a)-associated state transition (p K(a2)=8.56); this behavior differs from that of native cyt c, which shows a monophasic alkaline transition (p K(a)=8.9). The data indicate that the 40s Omega-loop plays an important role in the stability of cyt c and in ensuring a correct alkaline conformational transition of the protein.

Anions induce collapse of acid-denatured cytochrome c into a compact state, the A-state, showing molten globule character. Since structural information on partially folded forms of proteins is important for a deeper understanding of folding mechanisms and of the factors affecting protein stabilization, in this paper we have investigated in detail the effects of anions on the tertiary conformation of the A-state. We have found that the salt-induced collapse of acid-denatured cytochrome c leads to a number of equilibria between high-spin and low-spin heme states and between two types of low-spin states. The two latter states are characterized by conformations leading to a native-like Met-Fe-His axial coordination and a bis-His configuration. The equilibrium between these two A-states is dependent on the concentration and/or size of the anions (i.e. the bigger the anion, the greater its effect). Further, on the basis of fast kinetic data, a kinetic model of the folding process from the acid-unfolded protein to the A-state (at low and high anion concentration) is described.

Horseradish peroxidase isoenzyme C (HRPC) is one of the most intensively studied members of the peroxidase superfamily, a group of heme containing enzymes which oxidise a wide variety of aromatic molecules in the presence of hydrogen peroxide. The recent X-ray crystal structure of HRPC [1] has confirmed previous analyses of calcium content indicating 2 moles of calcium per mole of enzyme, one on either side of the heme. Apo-HRPC has been found not to properly refold in the absence of calcium ions [2] and removal of the bound calcium ions from native HRPC results in a considerable decrease in activity and thermal stability [3, 4]. The particular importance of the distal calcium ion for the catalytic activity has been demonstrated by mutation of distal residues of HRPC which are H-bonded with the distal Ca ion [5].

Heme-containing peroxidases from secretory plant peroxidases are classified in class III of the “plant peroxidases superfamily”, to distinguish them from intracellular peroxidases of prokariotic origin (class I) and secretory fungal peroxidases (class II) [1]. All these peroxidases have key residues conserved in the heme cavity which are important for the enzymatic activity [2].

The first quantitative characterization of the interaction of NO 2 - with the Cu-containing dissimilatory nitrite reductase (NiR) of Alcaligenes xylosoxidans using steady-state kinetics, equilibrium gel filtration and EPR spectroscopy is described. Each molecule of this protein consists of three equivalent subunits, each containing a type-1 Cu atom and also a type-2 Cu atom at each subunit interface. Enzyme activity increased in a biphasic manner with decreasing pH, having an optimum at pH 5.2 and a plateau between pH 6.1 and 5.8. Equilibrium gel filtration showed that binding of NO 2 - to the oxidized NiR was also pH-dependent. At pH 7.5, no binding was detectable, but binding was detectable at lower pH values. At pH 5.2, the concentration-dependence for binding of NO 2 - to the enzyme showed that approx. 4.1 NO 2 - ions bound per trimeric NiR molecule. Unexpectedly, NiR deficient in type-2 Cu centres bound 1.3 NO 2 - ions per trimer. When corrected for this binding, a value of 3 NO 2 - ions bound per trimer of NiR, equivalent to the type-2 Cu content. The NO 2 - -induced changes in the EPR parameters of the type-2 Cu centre of the oxidized enzyme showed a similar pH-dependence to that of the activity. Binding constants for NO 2 - at a single type of site, after allowing for the non-specifically bound NO 2 - , were 350±35 μ M (mean±S.E.M.) at pH 7.5 and μ M at pH 5.2. The apparent K m for NO 2 - with saturating concentrations of dithionite as reductant was 35 μ M at pH 7.5, which is 10-fold tighter than for the oxidized enzyme, and is compatible with an ordered mechanism in which the enzyme is reduced before NO 2 - binds.

Resonance Raman (RR) spectroscopy has been particularly valuable in providing information on the heme pocket of horseradish peroxidase (HRP-C) [1] and cytochrome c peroxidase (CCP) [2 and references therein]. Comparison of these studies suggested that the heme pocket structure is significantly different in the two proteins whereas the hydrogen-bonding network extending between the distal and proximal sides of the heme appears to be maintained. The RR studies showed that HRP-C is a mixture of 6-coordinate high spin (6-cHS) and 5-coordinate high spin (5-cHS) forms, with the 5-cHS as the dominant species [1]. It was also noted that some of the RR core size marker bands in HRP were about 10cm-1 higher than those normally observed for 5-cHS and 6-cHS hemes. They were referred to as 5-cHS# and 6-cHS# to distinguish them from ‘normal’ hemes. The differences are suggested to result from the HS heme in HRP-C having a different degree of distortion from planarity than other heme proteins. In the present study RR data from the recombinant HRP-C distal site mutant, His42→Leu (H42L), will be discussed and compared with other distal site mutants of HRP and the corresponding mutant (H52L) of CCP (3).

The reactions of barley peroxidase 1 (BP 1) with hydrogen peroxide and ferulic acid (FA), caffeic acid and coniferyl alcohol have previously been investigated. These studies showed that BP 1 is a very unusual peroxidase. Under transient state conditions, the reaction of BP 1 with hydrogen peroxide alone is pH dependent in a way opposite to the pH dependence of other plant peroxidases. The reactivity of BP 1 is strongly increased as the pH is decreased from pH 5 to 3. At pH values higher than 5, BP 1 is almost inactive [1]. Under steady state conditions, at pH 4 the reaction of BP 1 was shown to be 10–40 times enhanced by the presence of one of the three substrates mentioned above. The enhancement has been proposed to be due to binding of the substrates to the resting state enzyme and thereby giving a more active form of the enzyme. This is supported by the Kd for the BP 1-FA binding of 7 µM, which is a very strong binding of a substrate to a peroxidase [2].

The major haemoglobin of the sub-Antarctic fish Eleginops maclovinus was structurally and functionally characterised with the aim to compare molecular environmental adaptations in the O(2)-transport system of sub-Antarctic fishes of the suborder Notothenioidei with those of their high-latitude relatives. Ligand-binding kinetics of the major haemoglobin of E. maclovinus indicated strong stabilisation of the liganded quaternary T state, enhanced in the presence of the physiological allosteric effector ATP, compared to that of high-Antarctic Trematomus bernacchii. The activation enthalpy for O(2) dissociation was dramatically lower than that in T. bernacchii haemoglobin, suggesting remarkable differences in temperature sensitivity and structural changes associated with O(2) release and exit from the protein. The haemoglobin functional properties, together with the X-ray structure of the CO form at 1.49 Å resolution, the first of a temperate notothenioid, strongly support the hypothesis that in E. maclovinus, whose life-style varies according to changes in habitat, the mechanisms that regulate O(2) affinity and the ATP-induced Root effect differ from those of high-Antarctic Notothenioids.