Enrica Droghetti

Role of Lysines in Cytochrome c−Cardiolipin Interaction

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.

Role of the Main Access Channel of Catalase-Peroxidase in Catalysis

Catalase-peroxidases (KatG) are bifunctional heme peroxidases with an overwhelming catalatic activity. The structures show that the buried heme b is connected to the exterior of the enzyme by a main channel built up by KatG-specific loops named large loop LL1 and LL2, the former containing the highly conserved sequence Met-Gly-Leu-Ile-Tyr-Val-Asn-Pro-Glu-Gly. LL1 residues Ile248, Asn251, Pro252, and Glu253 of KatG from Synechocystis are the focus of this study because of their exposure to the solute matrix of the access channel. In particular, the I248F, N251L, P252A, E253Q, and E253D mutants have been analyzed by UV-visible and resonance Raman spectroscopies in combination with steady-state and presteady-state kinetic analyses. Exchange of these residues did not alter the kinetics of cyanide binding or the overall peroxidase activity. Moreover, the kinetics of compound I formation and reduction by one-electron donors was similar in the variants and the wild-type enzyme. However, the turnover numbers of the catalase activity of I248F, N251L, E253Q, and E253D were only 12.3, 32.6, 25, and 42% of the wild-type activity, respectively. These findings demonstrate that the oxidation reaction of hydrogen peroxide (not its reduction) was affected by these mutations. The altered kinetics allowed us to monitor the spectral features of the dominating redox intermediate of E253Q in the catalase cycle. Resonance Raman data and structural analysis demonstrated the existence of a very rigid and ordered structure built up by the interactions of these residues with distal side and also (via LL1) proximal side amino acids, with the heme itself, and with the solute matrix in the channel. The role of Glu253 and the other investigated channel residues in maintaining an ordered matrix of oriented water dipoles, which guides hydrogen peroxide to its site of oxidation, is discussed.

Fluoride as a probe for h-bonding interactions in the active site of heme proteins: The case of thermobifida fusca hemoglobin

The structural and functional properties of the active site of the bacterial hemoglobin from Thermobifida fusca are largely determined by three polar amino acids: TrpG8, TyrCD1, and TyrB10. We have exploited the availability of a combinatorial set of mutants, in each of which these three amino acids have been singly, doubly, or triply replaced by a Phe residue, to perform a detailed study on H-bonding interactions between the protein and heme-bound fluoride. By appropriate choice of the excitation conditions, ν(Fe-F) stretching bands have been detected in the resonance Raman spectra. In the wild-type protein and one of the mutants, two ν(Fe-F) bands have been observed and assigned to the presence of two protein conformers where fluoride is singly or doubly H-bonded. Furthermore, by plotting the CT1 charge-transfer transition energy vs the ν(Fe-F) wavenumbers, an empirical correlation has been found. The data are well fitted by a straight line with a positive slope. The position along the correlation line can be considered as a novel, general spectroscopic indicator of the extent of H-bonding in the active site of heme proteins. In agreement with the spectroscopic results, we have observed that the rate of ligand dissociation in stopped-flow kinetic measurements progressively increases upon substitution of the H-bonding amino acids. Molecular dynamics simulations have been performed on the fluoride complexes of native and mutated forms, indicating the prevalent interactions at the active site. All the techniques yield evidence that TrpG8 and TyrCD1 can form strong H bonds with fluoride, whereas TyrB10 plays only a minor role in the stabilization of the ligand.

Eukaryotic extracellular catalase–peroxidase from Magnaporthe grisea – Biophysical/chemical characterization of the first representative from a novel phytopathogenic KatG group

All phytopathogenic fungi have two catalase–peroxidase paralogues located either intracellularly (KatG1) or extracellularly (KatG2). Here, for the first time a secreted bifunctional, homodimeric catalase–peroxidase (KatG2 from the rice blast fungus Magnaporthe grisea) has been produced heterologously with almost 100% heme occupancy and comprehensively investigated by using a broad set of methods including UV–Vis, ECD and resonance Raman spectroscopy (RR), thin-layer spectroelectrochemistry, mass spectrometry, steady-state & presteady-state spectroscopy. RR spectroscopy reveals that MagKatG2 shows a unique mixed-spin state, non-planar heme b, and a proximal histidine with pronounced imidazolate character. At pH 7.0 and 25 °C, the standard reduction potential E°′ of the Fe(III)/Fe(II) couple for the high-spin native protein was found to fall in the range typical for the KatG family. Binding of cyanide was relatively slow at pH 7.0 and 25 °C and with a Kd value significantly higher than for the intracellular counterpart. Demonstrated by mass spectrometry MagKatG2 has the typical Trp118-Tyr251-Met277 adduct that is essential for its predominantly catalase activity at the unique acidic pH optimum. In addition, MagKatG2 acts as a versatile peroxidase using both one- and two-electron donors. Based on these data, structure–function relationships of extracellular eukaryotic KatGs are discussed with respect to intracellular KatGs and possible role(s) in host–pathogen interaction.

The effects of ATP and sodium chloride on the cytochrome c–cardiolipin interaction: The contrasting behavior of the horse heart and yeast proteins

In cells a portion of cytochrome c (cyt c) (15-20%) is tightly bound to cardiolipin (CL), one of the phospholipids constituting the mitochondrial membrane. The CL-bound protein, which has nonnative tertiary structure, altered heme pocket, and disrupted Fe(III)-M80 axial bond, is thought to play a role in the apoptotic process. This has attracted considerable interest in order to clarify the mechanisms governing the cyt c-CL interaction. Herein we have investigated the binding reaction of CL with the c-type cytochromes from horse heart and yeast. Although the two proteins possess a similar tertiary architecture, yeast cyt c displays lower stability and, contrary to the equine protein, it does not bind ATP and lacks pro-apoptotic activity. The study has been performed in the absence and in the presence of ATP and NaCl, two compounds that influence the (horse cyt c)-CL binding process and, thus, the pro-apoptotic activity of the protein. The two proteins behave differently: while CL interaction with horse cyt c is strongly influenced by the two effectors, no effect is observed for yeast cyt c. It is noteworthy that NaCl induces dissociation of the (horse cyt c)-CL complex but has no influence on that of yeast cyt c. The differences found for the two proteins highlight that specific structural factors, such as the different local structure conformation of the regions involved in the interactions with either CL or ATP, can significantly affect the behavior of cyt c in its reaction with liposomes and the subsequent pro-apoptotic action of the protein.

The optical spectra of fluoride complexes can effectively probe H-bonding interactions in the distal cavity of heme proteins

Fluoride complexes of heme proteins are characterized by unique spectroscopic properties, that provide a simple and direct means to monitor the interactions of the distal heme pocket environment with the iron-bound ligand. In particular, a strong correlation has been demonstrated between the wavelength of the iron-porphyrin charge transfer band at 600-620nm (CT1) and the strength of H-bonding donation from the distal amino acid side chains to the fluoride ion. In parallel, resonance Raman spectra with excitation within either the CT1 band or the charge transfer band at 450-460nm (CT2) have revealed that the iron-fluoride stretching frequency is directly affected by H-bonding to the fluoride ion. On this basis, globins and peroxidases display distinct spectroscopic features, which are strongly dependent on the capability of their distal residues (i.e. histidine, arginine and tryptophan) to be involved in H-bonding with the ligand. In particular, in peroxidases strong H-bonding corresponds to a low iron-fluoride stretching frequency and to a red-shifted CT1 band. The reverse is observed in myoglobin. Interestingly, a truncated hemoglobin of microbial origin (Thermobifida fusca) investigated in the present work, displays the specific spectroscopic signature of a peroxidase, in agreement with the presence of strong H-bonding residues, i.e., tyrosine and tryptophan, within the distal pocket.

Histidine E7 dynamics modulates ligand exchange between distal pocket and solvent in AHb1 from Arabidopsis thaliana.

The distal His residue in type 1 nonsymbiotic hemoglobin AHb1 from Arabidopsis thaliana plays a fundamental role in stabilizing the bound ligand. This residue might also be important in regulating the accessibility to the distal cavity. The feasibility of this functional role has been examined using a combination of experimental and computational methods. We show that the exchange of CO between the solvent and the reaction site is modulated by a swinging motion of the distal His, which opens a channel that connects directly the distal heme pocket with the solvent. The nearby PheB10 aids the distal His in the stabilization of the bound ligand by providing additional protection against solvation. Overall, these findings provide evidence supporting the functional implications of the conformational rearrangement found for the distal His in AHb1, which mimics the gating role proposed for the same residue in myoglobin.

H-bonding networks of the distal residues and water molecules in the active site of Thermobifida fusca hemoglobin ☆

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. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Functional and Spectroscopic Characterization of Chlamydomonas reinhardtii Truncated Hemoglobins.

The single-cell green alga Chlamydomonas reinhardtii harbors twelve truncated hemoglobins (Cr-TrHbs). Cr-TrHb1-1 and Cr-TrHb1-8 have been postulated to be parts of the nitrogen assimilation pathway, and of a NO-dependent signaling pathway, respectively. Here, spectroscopic and reactivity properties of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4, all belonging to clsss 1 (previously known as group N or group I), are reported. The ferric form of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 displays a stable 6cLS heme-Fe atom, whereas the hexa-coordination of the ferrous derivative appears less strongly stabilized. Accordingly, kinetics of azide binding to ferric Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are independent of the ligand concentration. Conversely, kinetics of CO or NO2 − binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are ligand-dependent at low CO or NO2 − concentrations, tending to level off at high ligand concentrations, suggesting the presence of a rate-limiting step. In agreement with the different heme-Fe environments, the pH-dependent kinetics for CO and NO2−binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are characterized by different ligand-linked protonation events. This raises the question of whether the simultaneous presence in C. reinhardtii of multiple TrHb1s may be related to different regulatory roles.

Interplay of the H-bond donor-acceptor role of the distal residues in hydroxyl ligand stabilization of Thermobifida fusca truncated hemoglobin

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.