Giampiero De Sanctis

Functional Modulation by Lactate of Myoglobin A MONOMERIC ALLOSTERIC HEMOPROTEIN

The effect of lactate on O2 binding properties of sperm whale and horse heart myoglobins (Mb) has been investigated at moderately acid pH (i.e. pH 6.5, a condition which may be achieved in vivo under a physical effort). Addition of lactate brings about a decrease of O2 affinity (i.e. an increase of P50) in sperm whale and horse heart myoglobins. Accordingly, lactate shows a different affinity for the deoxygenated and oxygenated form, behaving as a heterotropic modulator. The lactate effect on O2 affinity appears to differ for sperm whale and horse heart Mb, δlogP50 being ≈1.0 and ≈0.4, respectively. From the kinetic viewpoint, the variation of O2 affinity for both myoglobins can be attributed mainly to a decrease of the kinetic association rate constant for ligand binding.

The truncated hemoglobin from Mycobacterium leprae

Truncated hemoglobins (trHbs) form a family of low molecular weight O2 binding hemoproteins distributed in eubacteria, protozoa, and plants. TrHbs branch in a distinct clade within the hemoglobin (Hb) superfamily. A unique globin gene has recently been identified from the complete genome sequence of Mycobacterium leprae that is predicted to encode a trHb (M. leprae trHbO). Sequence comparison and modelling considerations indicate that monomeric M. leprae trHbO has structural features typical of trHbs, such as 20-40 fewer residues than conventional globin chains, Gly-based sequence consensus motifs, likely assembling into a 2-on-2 alpha-helical sandwich fold, and hydrophobic residues recognized to build up the protein matrix ligand diffusion tunnel. The ferrous heme iron atom of deoxygenated M. leprae trHbO appears to be hexacoordinated, like in Arabidopsis thaliana trHbO-3 (A. thaliana trHbO-3). Accordingly, the value of the second-order rate constant for M. leprae trHbO carbonylation (7.3 x 10(3) M(-1) s(-1)) is similar to that observed for A. thaliana trHbO-3 (1.4 x 10(4) M(-1) s(-1)) and turns out to be lower than that reported for carbon monoxide binding to pentacoordinated Mycobacterium tuberculosis trHbN (6.7 x 10(6) M(-1) s(-1)). The lower reactivity of M. leprae trHbO as compared to M. tuberculosis trHbN might be related to the higher susceptibility of the leprosy bacillus to toxic nitrogen and oxygen species produced by phagocytic cells.

Mini-myoglobin: the structural significance of haem-ligand interactions

The properties of purified mini-myoglobin, the fragment 32-139 of horse heart myoglobin reconstituted with protohaem, have been investigated from a structural and functional view point. The recovery of secondary structure observed in the carbon monoxide derivative of mini-myoglobin, as shown by circular dichroism, and the overall similarity of the haem pocket to that of myoglobin, as deduced from the fluorescence properties of the complex with 1-anilino-8-naphthalene sulphonate, indicate that, in the presence of the constraints imposed by the haem and its ligands, the miniprotein reacquires a conformation close to that of native myoglobin. These spectroscopic data parallel the conclusions drawn from the results of ligand combination and dissociation kinetics; stopped-flow experiments indicate that carbon monoxide and oxygen bind to mini-myoglobin with rates almost identical with those of myoglobin itself. The significance of mini-myoglobin as a model of an oxygen-carrying protein, with some of the expected functional characteristics of an ancestor haemoprotein, is discussed, with reference to the mosaic structure of the myoglobin gene and the role of different exons in the evolution of proteins.

Reductive nitrosylation of ferric human serum heme-albumin.

Heme endows human serum albumin (HSA) with heme‐protein‐like reactivity and spectroscopic properties. Here, the kinetics and thermodynamics of reductive nitrosylation of ferric human serum heme‐albumin [HSA‐heme‐Fe(III)] are reported. All data were obtained at 20 °C. At pH 5.5, HSA‐heme‐Fe(III) binds nitrogen monoxide (NO) reversibly, leading to the formation of nitrosylated HSA‐heme‐Fe(III) [HSA‐heme‐Fe(III)‐NO]. By contrast, at pH ≥ 6.5, the addition of NO to HSA‐heme‐Fe(III) leads to the transient formation of HSA‐heme‐Fe(III)‐NO in equilibrium with HSA‐heme‐Fe(II)‐NO+. Then, HSA‐heme‐Fe(II)‐NO+ undergoes nucleophilic attack by OH− to yield ferrous human serum heme‐albumin [HSA‐heme‐Fe(II)]. HSA‐heme‐Fe(II) further reacts with NO to give nitrosylated HSA‐heme‐Fe(II) [HSA‐heme‐Fe(II)‐NO]. The rate‐limiting step for reductive nitrosylation of HSA‐heme‐Fe(III) is represented by the OH−‐mediated reduction of HSA‐heme‐Fe(II)‐NO+ to HSA‐heme‐Fe(II). The value of the second‐order rate constant for OH−‐mediated reduction of HSA‐heme‐Fe(II)‐NO+ to HSA‐heme‐Fe(II) is 4.4 × 103 m−1·s−1. The present results highlight the role of HSA‐heme‐Fe in scavenging reactive nitrogen species.

Ibuprofen modulates allosterically NO dissociation from ferrous nitrosylated human serum heme-albumin by binding to three sites

Human serum albumin (HSA) is a monomeric allosteric protein. Here, the effect of ibuprofen on denitrosylation kinetics (k(off)) and spectroscopic properties of HSA-heme-Fe(II)-NO is reported. The k(off) value increases from (1.4+/-0.2)x10(-4)s(-1), in the absence of the drug, to (9.5+/-1.2)x10(-3)s(-1), in the presence of 1.0x10(-2)M ibuprofen, at pH 7.0 and 10.0 degrees C. From the dependence of k(off) on the drug concentration, values of the dissociation equilibrium constants for ibuprofen binding to HSA-heme-Fe(II)-NO (K(1)=(3.1+/-0.4)x10(-7)M, K(2)=(1.7+/-0.2)x10(-4)M, and K(3)=(2.2+/-0.2)x10(-3)M) were determined. The K(3) value corresponds to the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(II)-NO determined by monitoring drug-dependent absorbance spectroscopic changes (H=(2.6+/-0.3)x10(-3)M). Present data indicate that ibuprofen binds to the FA3-FA4 cleft (Sudlows site II), to the FA6 site, and possibly to the FA2 pocket, inducing the hexa-coordination of HSA-heme-Fe(II)-NO and triggering the heme-ligand dissociation kinetics.

Influence of glycerol on the structure and redox properties of horse heart cytochrome c. A circular dichroism and electrochemical study

The effect of glycerol on the structure and redox properties of horse heart cytochrome c was investigated by absorption spectroscopy, circular dichroism, and dc cyclic voltammetry techniques. The results show that the organic solvent increases the α-helix structure of the protein and induces slight changes at the active-site environment; however, the overall tertiary structure does not appear to be significantly perturbed. Glycerol stabilizes cytochrome c, the free energy of denaturation (ΔG0) being approximately 0.7 kcal/mol larger than that determined in phosphate buffer under the same conditions, and influences the heterogeneous electron transfer kinetics at a chemically modified gold electrode; on the other hand, the redox potential of the protein is unaltered. On the whole, the results obtained indicate that glycerol acts as a suitable stabilizing agent of cytochrome c, which is of interest for application in biotechnology; the organic solvent does not alter the tertiary structure significantly or the redox properties of the protein. This has to be interpreted not only in terms of the glycerol-induced solvent ordering around the protein surface, but also as due to the specific features of the protein matrix.

Reciprocal allosteric modulation of carbon monoxide and warfarin binding to ferrous human serum heme-albumin.

Human serum albumin (HSA), the most abundant protein in human plasma, could be considered as a prototypic monomeric allosteric protein, since the ligand-dependent conformational adaptability of HSA spreads beyond the immediate proximity of the binding site(s). As a matter of fact, HSA is a major transport protein in the bloodstream and the regulation of the functional allosteric interrelationships between the different binding sites represents a fundamental information for the knowledge of its transport function. Here, kinetics and thermodynamics of the allosteric modulation: (i) of carbon monoxide (CO) binding to ferrous human serum heme-albumin (HSA-heme-Fe(II)) by warfarin (WF), and (ii) of WF binding to HSA-heme-Fe(II) by CO are reported. All data were obtained at pH 7.0 and 25°C. Kinetics of CO and WF binding to the FA1 and FA7 sites of HSA-heme-Fe(II), respectively, follows a multi-exponential behavior (with the same relative percentage for the two ligands). This can be accounted for by the existence of multiple conformations and/or heme-protein axial coordination forms of HSA-heme-Fe(II). The HSA-heme-Fe(II) populations have been characterized by resonance Raman spectroscopy, indicating the coexistence of different species characterized by four-, five- and six-coordination of the heme-Fe atom. As a whole, these results suggest that: (i) upon CO binding a conformational change of HSA-heme-Fe(II) takes place (likely reflecting the displacement of an endogenous ligand by CO), and (ii) CO and/or WF binding brings about a ligand-dependent variation of the HSA-heme-Fe(II) population distribution of the various coordinating species. The detailed thermodynamic and kinetic analysis here reported allows a quantitative description of the mutual allosteric effect of CO and WF binding to HSA-heme-Fe(II).

Coupling of the Oxygen-linked Interaction Energy for Inositol Hexakisphosphate and Bezafibrate Binding to Human HbA0

The energetics of signal propagation between different functional domains (i.e. the binding sites for O2, inositol hexakisphospate (IHP), and bezafibrate (BZF)) of human HbA0 was analyzed at different heme ligation states and through the use of a stable, partially heme ligated intermediate. Present data allow three main conclusions to be drawn, and namely: (i) IHP and BZF enhance each others binding as the oxygenation proceeds, the coupling free energy going from close to zero in the deoxy state to −3.4 kJ/mol in the oxygenated form; (ii) the simultaneous presence of IHP and BZF stabilizes the hemoglobin T quaternary structure at very low O2 pressures, but as oxygenation proceeds it does not impair the transition toward the R structure, which indeed occurs also under these conditions; (iii) under room air pressure (i.e. pO2 = 150 torr), IHP and BZF together induce the formation of an asymmetric dioxygenated hemoglobin tetramer, whose features appear reminiscent of those suggested for transition state species (i.e. T- and R-like tertiary conformation(s) within a quaternary R-like structure).

Mini-myoglobin: Electron paramagnetic resonance and reversible oxygenation of the cobalt derivative

Mini-myoglobin, obtained by limited proteolysis of horse heart myoglobin (residues 32 to 139), represents a good model for testing the correlation between an exon and a protein domain. We have shown that ligand binding kinetics, spectral and folding features of mini-myoglobin are very similar to those of native myoglobin. In order to develop further the analysis of the structure-function relationship in this mini-protein, mini-globin was reconstituted with the heme moiety in which iron is replaced by cobalt. The Soret absorption spectra of oxy and deoxy cobaltous mini-myoglobin are very similar to those of cobaltous myoglobin derivatives; in addition. Co-mini-myoglobin binds oxygen reversibly with an n value approximately 1 and a p50 value of 45 to 50 mm Hg (the same as Co-myoglobin). Oxy Co-mini-myoglobin shows a well-resolved electron paramagnetic resonance (e.p.r.) spectrum typical of an oxygenated hemoprotein, while the spectrum of the deoxy derivative, although similar to that of deoxy Co-myoglobin, displays a lower resolution of the complex hyperfine structure. Moreover, photodissociation experiments on oxy Co-mini-myoglobin allow e.p.r. detection of an intermediate state, already observed in most hemoproteins and diagnostic for the interaction of bound oxygen with the distal histidine residue. Thus, reconstitution of mini-globin with cobalt protoprophyrin IX has provided, for the first time, a stable oxygenated complex that reflects a correct folding of the protein surrounding the heme pocket and possesses the functional behaviour typical of a hemoprotein.

Proton Linkage for CO Binding and Redox Properties of Bovine Lactoperoxidase

The pH-dependence of redox properties and of CO binding to bovine lactoperoxidase has been investigated over the range between 2 and 11. The pH-dependence of redox potentials shows a biphasic behavior, suggesting the existence of (at least) two redox-linked groups, which change their pKa values upon reduction. These values are in close agreement with those observed to play a relevant role in the modulation of CO binding to ferrous bovine lactoperoxidase. They have been tentatively attributed to Arg-372 and His-226, which are located on the distal side of the heme pocket of lactoperoxidase. A complete and unequivocal description of the proton-linked behavior of bovine lactoperoxidase requires, however, three residues, which are redox linked and relevant for the modulation of CO binding. The rate constant for CO binding to bovine lactoperoxidase is slower than what is reported for most hemoproteins, suggesting that these two residues, Arg-372 and His-226, are representing a severe barrier for the access of exogenous ligands to the heme. This aspect has been further investigated by fast kinetics following laser photolysis, trying to obtain information on the ligand binding pathway and on the energy barriers.