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Structural and functional properties of ferrous Mycobacterium tuberculosis ( Mt -Nb) and human ( Hs -Nb) nitrobindins (Nbs) were investigated. At pH 7.0 and 25.0 °C, the unliganded Fe(II) species is penta-coordinated and unlike most other hemoproteins no pH-dependence of its coordination was detected over the pH range between 2.2 and 7.0. Further, despite a very open distal side of the heme pocket (as also indicated by the vanishingly small geminate recombination of CO for both Nbs), which exposes the heme pocket to the bulk solvent, their reactivity toward ligands, such as CO and NO, is significantly slower than in most hemoproteins, envisaging either a proximal barrier for ligand binding and/or crowding of H 2 O molecules in the distal side of the heme pocket which impairs ligand binding to the heme Fe-atom. On the other hand, liganded species display already at pH 7.0 and 25 °C a severe weakening (in the case of CO) and a cleavage (in the case of NO) of the proximal Fe-His bond, suggesting that the ligand-linked movement of the Fe(II) atom onto the heme plane brings about a marked lengthening of the proximal Fe-imidazole bond, eventually leading to its rupture. This structural evidence is accompanied by a marked enhancement of both ligands dissociation rate constants. As a whole, these data clearly indicate that structural-functional relationships in Nbs strongly differ from what observed in mammalian and truncated hemoproteins, suggesting that Nbs play a functional role clearly distinct from other eukaryotic and prokaryotic hemoproteins.

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., (© 2018 Wiley Periodicals, Inc.)

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.

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).

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand binding capacity. HSA participates in heme scavenging by binding the macrocycle at fatty acid site 1. In turn, heme endows HSA with globin-like reactivity and spectroscopic properties. A detailed pH-dependent kinetic and spectroscopic investigation of iron(II) heme-HSA and of its carbonylated form is reported here. Iron (II) heme-HSA is a mixture of a four-coordinate intermediate-spin species (predominant at pH 5.8 and 7.0), a five-coordinate high-spin form (mainly at pH 7.0), and a six-coordinate low-spin species (predominant at pH 10.0). The acidic-to-alkaline reversible transition reflects conformational changes leading to the coordination of the heme Fe(II) atom by the His146 residue via its nitrogen atom, both in the presence and in the absence of CO. The presence of several species accounts for the complex, multiexponential kinetics observed and reflects the very slow interconversion between the different species observed both for CO association to the free iron(II) heme-HSA and for CO dissociation from CO-iron(II) heme-HSA as a function of pH., (© SBIC 2011)

A robust and dependable headspace HS-GC-MS method has been developed for the determination of carbon monoxide (CO) treated tuna fish. The performance of the method has been evaluated by analyzing spiked and blank samples. The proposed method uses a programmed temperature vaporizing (PTV) injector to implement the HS injection and overcome the negative effects on the column of water contained in the sample. The level of CO could be easily detected in the treated samples, its amount differing markedly from that detected in the untreated samples. Proper oven temperature programming prevented any deterioration of chromatographic performance caused by the accumulation of water, carbon dioxide and other low molecular weight hydrocarbons in the column during serial analyses. The introduction of PTV injection and the restoring of GC column, by oven temperature programming, were crucial for obtaining the required robustness. This procedure assures the run-to-run repeatability necessary for high throughput applications. The method affords linear calibration curves ranging from 50 to 2500 ng/g of CO content in tuna sample., (2010 John Wiley & Sons, Ltd.)

The active site of the oxygen-avid truncated hemoglobin from Bacillus subtilis has been characterized by infrared absorption and resonance Raman spectroscopies, and the dynamics of CO rebinding after photolysis has been investigated by picosecond transient absorption spectroscopy. Resonance Raman experiments on the CO bound adduct revealed the presence of two Fe-CO stretching bands at 545 and 520 cm-1, respectively. Accordingly, two C-O stretching bands at 1924 and 1888 cm-1 were observed in infrared absorption and resonance Raman measurements. The very low C-O stretching frequency at 1888 cm-1 (corresponding to the extremely high RR stretching frequency at 545 cm-1) indicates unusually strong hydrogen bonding between CO and distal residues. On the basis of a comparison with other truncated hemoglobin it is envisaged that the two CO conformers are determined by specific interactions with the TrpG8 and TyrB10 residues. Mutation of TrpG8 to Leu deeply alters the hydrogen-bonding network giving rise mainly to a CO conformer characterized by a Fe-CO stretching band at 489 cm-1 and a CO stretching band at 1958 cm-1. Picosecond laser photolysis experiments carried out on the CO bound adduct revealed dynamical processes that take place within a few nanoseconds after photolysis. Picosecond dynamics is largely dominated by CO geminate rebinding and is consistent with strong H-bonding contributions of TyrB10 and TrpG8 to ligand stabilization.

The nonsymbiotic hemoglobins, AHb1 and AHb2, have recently been isolated from Arabidopsis thaliana. Using steady-state and time-resolved spectroscopic methods, we show that Fe2+ AHb1 contains a mixture of penta- and hexacoordinated heme, while Fe2+ AHb2 is fully hexacoordinated. In the CO complexes, polar interactions and H-bonds with the ligand are stronger for AHb1 than for AHb2. The ligand binding kinetics are substantially different, reflecting the distribution between the penta- and hexacoordinated species, and indicate that protein dynamics and ligand migration pathways are very specific for each of the two proteins. In particular, a very small, non-exponential geminate rebinding observed in AHb1 suggests that the distal heme cavity is connected with the exterior by a relatively open channel. The large, temperature-dependent geminate rebinding observed for AHb2 implies a major role of protein dynamics in the ligand migration from the distal cavity to the solvent. The structures of AHb1 and AHb2, modeled on the basis of the homologous rice hemoglobin, exhibit a different cavity system that is fully compatible with the observed ligand binding kinetics. Overall, these kinetic and structural data are consistent with the putative NO-dioxygenase activity previously attributed to AHb1, whereas the role of AHb2 remains elusive.

Resonance Raman and infrared spectra and the CO dissociation rates (k(off)) were measured in Coprinus cinereus peroxidase (CIP) and several mutants in the heme binding pocket. These mutants included the Asp245Asn, Arg51Leu, Arg51Gln, Arg51Asn, Arg51Lys, Phe54Trp, and Phe54Val mutants. Binding of CO to CIP produced different CO adducts at pH 6 and 10. At pH 6, the bound CO is H-bonded to the protonated distal His55 residue, whereas at alkaline pH, the vibrational signatures and the rate of CO dissociation indicate a distal side which is more open or flexible than in other plant peroxidases. The distal Arg51 residue is important in determining the rate of dissociation in the acid form, increasing by 8-17-fold in the Arg51 mutants compared to that for the wild-type protein. Replacement of the distal Phe with Trp created a new acid form characterized by vibrational frequencies and k(off) values very similar to those of cytochrome c peroxidase.

CO binding kinetics to the homodimeric myoglobin (Mb) from Nassa mutabilis has been investigated between pH 1.9 and 7.0. Protonation of the proximal imidazole at low pH (less than or equal to 3.0) and the consequent cleavage of the HisF8NE2-Fe proximal bond brings about a approximately 20-fold increase of the second-order rate constant for CO binding. This process displays a pKa = 4.0 +/- 0.2, significantly higher than that observed in all other deoxygenated hemoproteins investigated up to now. Such a feature underlies a decreased energy for the HisF8NE2-Fe proximal bond in the unliganded form and it also appears supported by resonance Raman spectroscopy in the low frequency region of the Fe(II) deoxygenated hemoprotein. Further, the pH-rate profile of N. mutabilis Mb, like that of the homodimeric hemoglobin (Hb) from Scapharca inaequivalvis (Coletta, M., Boffi, A., Ascenzi, P., Brunori, M. and Chiancone, E. (1990) J. Biol. Chem. 265, 4828-4830), can be described only by assuming a concerted proton-linked transition with n = 1.8 +/- 0.1. Such a characteristic suggests, also on the basis of the amino acid sequence homology between N. mutabilis Mb and S. inaequivalvis Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different from that operative in other hemoproteins.

To investigate the molecular basis for the 100-fold slower rate of CO dissociation in ferrous peroxidases relative to myoglobin, CO dissociation rates were measured as a function of pH in the cloned cytochrome c peroxidase from yeast [CCP(MI)] and in several mutants in the heme binding pocket prepared by site-directed mutagenesis. The mutants included Asp 235----Asn; Arg 48----Lys, Leu; and His 181----Gly. Changes in the absorption spectrum with pH are consistent with conversion of the CO-ferrous CCP(MI) complex from acidic to alkaline forms by a two-proton cooperative ionization, with an apparent pKa = 7.6, analogous to that described for CCP from bakers' yeast [Iizuka, T., Makino, R., Ishimura, Y., & Yonetani, T. (1985) J. Biol. Chem. 260, 1407-1412]. The rate of CO dissociation (koff) was increased 11-fold (from 0.7 x 10(-4) to 8.0 x 10(-4) s-1) by conversion of the acidic to the alkaline form. Analogous acidic and alkaline forms of the CO complex were also observed in the mutants of CCP(MI) examined here. In the acidic form, koff was increased 5- and 20-fold when Arg 48 was replaced with Lys and Leu, respectively, while in the acidic form of mutants that possess Arg 48, koff was similar to that observed in CCP(MI). Conversion of the CO complex from the acidic to alkaline form increased koff in all the mutants, and the pH-dependent increase in koff correlated with a two-proton cooperative ionization, except in the case of His 181----Gly. In this mutant, pH-dependent increase in koff correlated with a single-proton ionization, implicating His 181 as one of the two residues that is deprotonated in the conversion of CO-ferrous CCP(MI) from acidic to alkaline forms. Only a 2.5-fold variation was observed for koff between the alkaline form of CCP(MI) and the Arg 48----Leu mutant, suggesting that the influence of Arg 48 on the rate of CO dissociation is decreased in the alkaline form by a conformational change.(ABSTRACT TRUNCATED AT 400 WORDS)

Vibrational frequencies associated with FeC and CO stretching and FeCO bending modes have been determined via resonance Raman (RR) and infrared (IR) spectroscopy for cytochrome c peroxidase (CCP) mutants prepared by site-directed mutagenesis. These include the bacterial "wild type", CCP(MI), and mutations involving groups on the proximal (Asp-235----Asn; Trp-191---Phe) and distal (Trp-51----Phe; Arg-48----Leu and Lys) side of the heme. The data were analyzed with the aid of a recently established correlation between nu FeC and nu CO, which can be used to distinguish between back-bonding and axial ligand donor effects. At high pH all adducts showed essentially the same vibrational pattern (form I') with nu FeC approximately 505 cm-1, nu CO approximately 1948 cm-1, and delta FeCO (weak RR band) approximately 576 cm-1. These frequencies are very similar to those shown by the myoglobin CO adduct and imply a "normal" H-bond of the proximal histidine. At pH 7 (pH 6 for Asn-235 and Leu-48), different forms are seen for different proteins: form I (nu FeC approximately 500 cm-1, nu CO = 1922-1941 cm-1, and delta FeCO approximately 580 cm-1, very weak) in the case of CCP(MI) and Phe-191, as well as bakers' yeast CCP, or form II (nu FeC approximately 530 cm-1, nu CO = 1922-1933 cm-1, and delta FeCO = 585 cm-1, moderately strong) for Asn-235 and Phe-51.(ABSTRACT TRUNCATED AT 250 WORDS)

Resonance Raman (RR) spectra are reported for CO-bound cytochrome c peroxidase (CCP). At low pH, two forms are observed: form II, with nu Fe-C = 530 cm-1 and delta FeCO = 585 cm-1, and form I, with nu Fe-C = 495 cm-1 and no detectable delta FeCO. They appear to have coincident nu CO infrared bands, at 1922 cm-1. These low-pH forms, similar to those observed for horseradish peroxidase (HRP), are attributed to tilted, H-bonded CO and perpendicular CO, respectively. The frequencies differ between the two proteins, a weaker H bond to CO being indicated for CCP. As with HRP, the equilibrium between forms I and II is shifted toward the latter at increasing CO concentrations, suggesting that secondary binding of CO perturbs the distal residues. At high pH [8.4, tris(hydroxymethyl)aminomethane buffer] the form II fraction converts to another form, II', with nu FeC = 503 cm-1, delta FeCO = 575 cm-1, and nu CO = 1948 cm-1; a tilted, non-H-bonded geometry is suggested. If phosphate buffer is used, however, form II (H bonded) persists at pH 8.4. This result establishes a role for phosphate in stabilizing the H-bonded form of the enzyme; it is suggested that phosphate binds near the distal imidazole and substantially increases its pKa. The conformational state is also influenced by aging. Fresh protein contains purely high spin FeIII heme, as monitored by the high-frequency RR spectrum, and yields form II almost exclusively at elevated CO concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Resonance Raman (RR) spectroscopy and infrared spectroscopy have been used to characterize the three vibrational modes, CO and FeC stretching and FeCO bending, for carbon monoxide bound to reduced horseradish peroxidase, with the aid of 13CO and C18O isotope shifts. At high pH, one species, I, is observed, with nu FeC = 490 cm-1 and nu CO = 1932 cm-1. The absence of a band attributable to delta FeCO suggests a linear FeCO unit normal to the heme plane. The data were consistent with I having a strongly H-bonded proximal histidine, as shown by a comparison with imidazole and imidazolate adducts of FeIIPPDME(CO) (PPDME = protoporphyrin IX dimethyl ester), with nu FeC = 497 and 492 cm-1 and nu CO = 1960 and 1942 cm-1. At low pH an additional species, II, is observed, with nu FeC = 537 cm-1, nu CO = 1904 cm-1, and delta FeCO = 587 cm-1; it is attributed to FeCO that is H bonded to a protonated distal histidine, the H bond strongly lowering nu CO and raising nu FeC. The appearance of delta FeCO in the RR spectrum suggests that the FeCO unit in II is tilted with respect to the heme plane. At low pH, the population of I and II depends on the CO concentration. I dominates at low CO/protein levels but is replaced by II as the amount of CO is increased. This behavior is suggested to arise from secondary binding of CO, which induces a conformation change involving the distal residues of the heme pocket.