Cyanide binding to truncated hemoglobins: a crystallographic and kinetic study

Cyanide is one of the few diatomic ligands able to interact with the ferric and ferrous heme-Fe atom. Here, the X-ray crystal structure of the cyanide derivative of ferric Mycobacterium tuberculosis truncated hemoglobin-N (M. tuberculosis trHbN) has been determined at 2.0 A (R-general = 17.8% and R-free = 23.5%), and analyzed in parallel with those of M. tuberculosis truncated hemoglobin-O (M. tuberculosis trHbO), Chlamydomonas eugametos truncated hemoglobin (C. eugametos trHb), and sperm whale myoglobin, generally taken as a molecular model. Cyanide binding to M. tuberculosis trHbN is stabilized directly by residue TyrB10(33), which may assist the deprotonation of the incoming ligand and the protonation of the outcoming cyanide. In M. tuberculosis trHbO and in C. eugametos trHb the ligand is stabilized by the distal pocket residues TyrCD1(36) and TrpG8(88), and by the TyrB10(20) - GlnE7(41) - GlnE11(45) triad, respectively. Moreover, kinetics for cyanide binding to ferric M. tuberculosis trHbN and trHbO and C. eugametos trHb, for ligand dissociation from the ferrous trHbs, and for the reduction of the heme-Fe(III)-cyanide complex have been determined, at pH 7.0 and 20.0 degrees C. Despite the different heme distal site structures and ligand interactions, values of the rate constant for cyanide binding to ferric (non)vertebrate heme proteins are similar, being influenced mainly by the presence in the heme pocket of proton acceptor group(s), whose function is to assist the deprotonation of the incoming ligand (i.e., HCN). On the other hand, values of the rate constant for the reduction of the heme-Fe(III)-cyanide (non)vertebrate globins span over several orders of magnitude, reflecting the different ability of the heme proteins considered to give productive complex(es) with dithionite or its reducing species SO(2)(-). Furthermore, values of the rate constant for ligand dissociation from heme-Fe(II)-cyanide (non)vertebrate heme proteins are very different, reflecting the different nature and geometry of the heme distal residue(s) hydrogen-bonded to the heme-bound cyanide.     

Peroxidase Activity and Involvement in the Oxidative Stress Response of Roseobacter denitrificans Truncated Hemoglobin

Roseobacter denitrificans is a member of the widespread marine Roseobacter genus. We report the first characterization of a truncated hemoglobin from R. denitrificans (Rd. trHb) that was purified in the heme-bound form from heterologous expression of the protein in Escherichia coli. Rd. trHb exhibits predominantly alpha-helical secondary structure and absorbs light at 412, 538 and 572 nm. The phylogenetic classification suggests that Rd. trHb falls into group II trHbs, whereas sequence alignments indicate that it shares certain important heme pocket residues with group I trHbs in addition to those of group II trHbs. The resonance Raman spectra indicate that the isolated Rd. trHb contains a ferric heme that is mostly 6-coordinate low-spin and that the heme of the ferrous form displays a mixture of 5- and 6-coordinate states. Two Fe-His stretching modes were detected, notably one at 248 cm^-1, which has been reported in peroxidases and some flavohemoglobins that contain an Fe-His-Asp (or Glu) catalytic triad, but was never reported before in a trHb. We show that Rd. trHb exhibits a significant peroxidase activity with a (k_cat/K_m) value three orders of magnitude higher than that of bovine Hb and only one order lower than that of horseradish peroxidase. This enzymatic activity is pH-dependent with a pK_a value ~6.8. Homology modeling suggests that residues known to be important for interactions with heme-bound ligands in group II trHbs from Mycobacterium tuberculosis and Bacillus subtilis are pointing toward to heme in Rd. trHb. Genomic organization and gene expression profiles imply possible functions for detoxification of reactive oxygen and nitrogen species in vivo. Altogether, Rd. trHb exhibits some distinctive features and appears equipped to help the bacterium to cope with reactive oxygen/nitrogen species and/or to operate redox biochemistry.     

Truncated hemoglobins: trimming the classical ‘three-over-three’ globin fold to a minimal size

Truncated hemoglobins (trHbs) host the heme in a “two-over-two’ α-helical sandwich which results from extensive editing of the classical ‘three-over-three’ globin fold. The three-dimensional structure of trHbs is based on four main α-helices, arranged in a sort of α-helical bundle composed of two antiparallel helix pairs (B/E and G/H). Most notably, trHbs deviate from the conventional globin fold in that they display an extended loop substituting for the heme proximal F-helix observed in globins. Moreover, since efficient adaptation of a 110–130 amino acid trHb chain to host the porphyrin ring firstly requires specific chain flexibility, trHbs contain three invariant Gly-based motifs. Inspection of the trHb three-dimensional trHb structures shows that an apparent protein cavity or tunnel would connect the protein surface to an inner region very close to the heme distal site. Such a structural feature, never observed before in (non) vertebrate globins, may have substantial implications for ligand diffusion and binding properties in trHbs.     

Ligand migration in the truncated hemoglobin of Mycobacterium tuberculosis

The truncated hemoglobin of Mycobacterium tuberculosis (Mt-trHbO) is a small heme protein belonging to the hemoglobin superfamily. Truncated hemoglobins (trHbs) are believed to have functional roles such as terminal oxidases and oxygen sensors involved in the response to oxidative and nitrosative stress, nitric oxide (NO) detoxification, O?/NO chemistry, O? delivery under hypoxic conditions, and long-term ligand storage. Based on sequence similarities, they are classified into three groups. Experimental studies revealed that all trHbs display a 2-on-2 α-helical sandwich fold rather than the 3-on-3 α-helical sandwich fold of the classical hemoglobin fold. Using locally enhanced sampling (LESMD) molecular dynamics, the ligand-binding escape pathways from the distal heme binding cavity of Mt-trHbO were determined to better understand how this protein functions. The importance of specific residues, such as the group II and III invariant W(G8) residue, can be seen in terms of ligand diffusion pathways and ligand dynamics. LESMD simulations show that the wild-type Mt-trHbO has three diffusion pathways while the W(G8)F Mt-trHbO mutant has only two. The W(G8) residue plays a critical role in ligand binding and stabilization and helps regulate the rate of ligand escape from the distal heme pocket. Thus, this invariant residue is important in creating ligand diffusion pathways and possibly in the enzymatic functions of this protein.     

Structural bases for heme binding and diatomic ligand recognition in truncated hemoglobins

Truncated hemoglobins (trHbs) are low-molecular-weight oxygen-binding heme-proteins distributed in eubacteria, cyanobacteria, unicellular eukaryotes, and in higher plants, constituting a distinct group within the hemoglobin (Hb) superfamily. TrHbs display amino acid sequences 20-40 residues shorter than classical (non)vertebrate Hbs and myoglobins, to which they are scarcely related by sequence similarity. The trHb tertiary structure is based on a 2-on-2 alpha-helical sandwich, which represents a striking editing of the highly conserved 3-on-3 alpha-helical globin fold, achieved through deletion/truncation of alpha-helices and specific residue substitutions. Despite their 'minimal' polypeptide chain span, trHbs display an inner tunnel/cavity system held to support ligand diffusion to/from the heme distal pocket, accumulation of heme ligands within the protein matrix, and/or multiligand reactions. Moreover, trHbs bind and effectively stabilize the heme and recognize diatomic ligands (i.e., O2, CO, NO, and cyanide), albeit with varying thermodynamic and kinetic parameters. Here, structural bases for heme binding and diatomic ligand recognition by trHbs are reviewed.     

Isoniazid Inhibits the Heme-Based Reactivity of Mycobacterium tuberculosis Truncated Hemoglobin N

Isoniazid represents a first-line anti-tuberculosis medication in prevention and treatment. This prodrug is activated by a mycobacterial catalase-peroxidase enzyme called KatG in Mycobacterium tuberculosis), thereby inhibiting the synthesis of mycolic acid, required for the mycobacterial cell wall. Moreover, isoniazid activation by KatG produces some radical species (e.g., nitrogen monoxide), that display anti-mycobacterial activity. Remarkably, the ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of (pseudo-)enzymatic detoxification systems, including truncated hemoglobins (trHbs). Here, we report that isoniazid binds reversibly to ferric and ferrous M. tuberculosis trHb type N (or group I; Mt-trHbN(III) and Mt-trHbN(II), respectively) with a simple bimolecular process, which perturbs the heme-based spectroscopic properties. Values of thermodynamic and kinetic parameters for isoniazid binding to Mt-trHbN(III) and Mt-trHbN(II) are K = (1.1±0.1)×10⁻⁴ M, k _on = (5.3±0.6)×10³ M⁻¹ s⁻¹ and k _off = (4.6±0.5)×10⁻¹ s⁻¹; and D = (1.2±0.2)×10⁻³ M, d _on = (1.3±0.4)×10³ M⁻¹ s⁻¹, and d _off = 1.5±0.4 s⁻¹, respectively, at pH 7.0 and 20.0°C. Accordingly, isoniazid inhibits competitively azide binding to Mt-trHbN(III) and Mt-trHbN(III)-catalyzed peroxynitrite isomerization. Moreover, isoniazid inhibits Mt-trHbN(II) oxygenation and carbonylation. Although the structure of the Mt-trHbN-isoniazid complex is not available, here we show by docking simulation that isoniazid binding to the heme-Fe atom indeed may take place. These data suggest a direct role of isoniazid to impair fundamental functions of mycobacteria, e.g. scavenging of reactive nitrogen and oxygen species, and metabolism.     

The pH dependence of the stereochemistry around the heme group in NO–cytochrome c (horse heart)

The electronic absorption, EPR and MCD spectra of NO derivatives of both ferrous and ferric cytochrome c (horse heart) have been measured in the pH region 2.0 to 12.9, in order to elucidate the pH dependence of the stereochemistry around the heme group. The reaction products of NO with ferrous cytochrome c in equilibrium were as follows: in the region 2.0 ⩽ pH ⩽ 5.3, NO–ferrous cytochrome c; in the region 5.3 < pH ⩽ 11.0, a mixture of NO–ferrous cytochrome c and native ferrous cytochrome c; at pH 12.0, NO–ferrous cytochrome c. At pH 2.0, the NO–ferrous cytochrome c contained a five-coordinate nitrosylheme as the major component and a six-coordinate species as the minor component, and at the order pH values it contained only the six-coordinate species. The reaction products of NO with ferric cytochrome c in equilibrium were as follows: in the region 2.0 ⩽ pH ⩽ 7.2, NO–ferric cytochrome c with six-coordinate nitrosylheme; in the region 7.2 < pH ⩽ 11.0, a mixture of NO–ferrous cytochrome c and native ferrous cytochrome c; at pH 12.0, NO–ferrous cytochrome c. Thus, the reaction of NO with ferric cytochrome c results in the formation of NO–ferrous cytochrome c, which is a typical case of reductive nitrosylation.     

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.     

Structural determinants in the group III truncated hemoglobin from Campylobacter jejuni

Truncated hemoglobins (trHbs) constitute a distinct lineage in the globin superfamily, distantly related in size and fold to myoglobin and monomeric hemoglobins. Their phylogenetic analyses revealed that three groups (I, II, and III) compose the trHb family. Group I and II trHbs adopt a simplified globin fold, essentially composed of a 2-on-2 alpha-helical sandwich, wrapped around the heme group. So far no structural data have been reported for group III trHbs. Here we report the three-dimensional structure of the group III trHbP from the eubacterium Campylobacter jejuni. The 2.15-A resolution crystal structure of C. jejuni trHbP (cyano-met form) shows that the 2-on-2 trHb fold is substantially conserved in the trHb group III, despite the absence of the Gly-based sequence motifs that were considered necessary for the attainment of the trHb specific fold. The heme crevice presents important structural modifications in the C-E region and in the FG helical hinge, with novel surface clefts at the proximal heme site. Contrary to what has been observed for group I and II trHbs, no protein matrix tunnel/cavity system is evident in C. jejuni trHbP. A gating movement of His(E7) side chain (found in two alternate conformations in the crystal structure) may be instrumental for ligand entry to the heme distal site. Sequence conservation allows extrapolating part of the structural results here reported to the whole trHb group III.     

Heme-assisted S-Nitrosation Desensitizes Ferric Soluble Guanylate Cyclase to Nitric Oxide

Nitric oxide (NO) signaling regulates key processes in cardiovascular physiology, specifically vasodilation, platelet aggregation, and leukocyte rolling. Soluble guanylate cyclase (sGC), the mammalian NO sensor, transduces an NO signal into the classical second messenger cyclic GMP (cGMP). NO binds to the ferrous (Fe²⁺) oxidation state of the sGC heme cofactor and stimulates formation of cGMP several hundred-fold. Oxidation of the sGC heme to the ferric (Fe³⁺) state desensitizes the enzyme to NO. The heme-oxidized state of sGC has emerged as a potential therapeutic target in the treatment of cardiovascular disease. Here, we investigate the molecular mechanism of NO desensitization and find that sGC undergoes a reductive nitrosylation reaction that is coupled to the S-nitrosation of sGC cysteines. We further characterize the kinetics of NO desensitization and find that heme-assisted nitrosothiol formation of β1Cys-78 and β1Cys-122 causes the NO desensitization of ferric sGC. Finally, we provide evidence that the mechanism of reductive nitrosylation is gated by a conformational change of the protein. These results yield insights into the function and dysfunction of sGC in cardiovascular disease.     

Electron paramagnetic resonance spectroscopy of lactoperoxidase complexes: clarification of hyperfine splitting for the NO adduct of lactoperoxidase

Electron paramagnetic resonance (EPR) studies of the nitrosyl adduct of ferrous lactoperoxidase (LPO) confirm that the fifth axial ligand in LPO is bound to the iron via a nitrogen atom. Complete reduction of the ferric LPO sample is required in order to observe the nine-line hyperfine splitting in the ferrous LPO/NO EPR spectrum. The ferrous LPO/NO complex does not exhibit a pH or buffer system dependence when examined by EPR. Interconversion of the ferrous LPO/NO complex and the ferric LPO/NO2- complex is achieved by addition of the appropriate oxidizing or reducing agent. Characterization of the low-spin LPO/NO2- complex by EPR and visible spectroscopy is reported. The pH dependence of the EPR spectra of ferric LPO and ferric LPO/CN- suggests that a high-spin anisotropic LPO complex is formed at high pH and an acid-alkaline transition of the protein conformation near the heme site does occur in LPO/CN-. The effect of tris(hydroxymethyl)aminomethane buffer on the LPO EPR spectrum is also examined.     

Unraveling the molecular basis for ligand binding in truncated hemoglobins: The trHbO Bacillus subtilis case

Truncated hemoglobins (trHbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. Their tertiary structure consists in a 2‐over‐2 helical sandwich, which display typically an inner tunnel/cavity system for ligand migration and/or storage. The microorganism Bacillus subtilis contains a peculiar trHb, which does not show an evident tunnel/cavity system connecting the protein active site with the solvent, and exhibits anyway a very high oxygen association rate. Moreover, resonant Raman results of CO bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. To understand these experimental results with atomistic detail, we performed classical molecular dynamics simulations of the oxy, carboxy, and deoxy proteins. The free energy profiles for ligand migration suggest that there is a key residue, GlnE11, that presents an alternate conformation, in which a wide ligand migration tunnel is formed, consistently with the kinetic data. This tunnel is topologically related to the one found in group I trHbs. On the other hand, the results for the CO and O₂ bound protein show that GlnE11 is directly involved in the stabilization of the cordinated ligand, playing a similar role as TyrB10 and TrpG8 in other trHbs. Our results not only reconcile the structural data with the kinetic information, but also provide additional insight into the general behaviour of trHbs. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Reversible hexa- to penta-coordination of the heme Fe atom modulates ligand binding properties of neuroglobin and cytoglobin

Neuroglobin (Ngb) and cytoglobin (Cygb) are two recently discovered intracellular members of the vertebrate hemoglobin (Hb) family. Ngb, predominantly expressed in nerve cells, is of ancient evolutionary origin and is homologous to nerve-globins of invertebrates. Cygb, present in many different tissues, shares common ancestry with myoglobin (Mb) and can be traced to early vertebrate evolution. Ngb is held to facilitate O2 diffusion to the mitochondria and to protect neuronal cells from hypoxic-ischemic insults, may be an oxidative stress-responsive sensor protein for signal transduction, and may carry out enzymatic activities, such as NO/O2 scavenging. Cygb is linked to collagen synthesis, may provide O2 for enzymatic reactions, and may be involved in a ROS(NO)-signaling pathway(s). Ngb and Cgb display the classical three-over-three alpha-helical fold of Hb and Mb, and are endowed with a hexa-coordinate heme-Fe atom, in their ferrous and ferric forms, having the heme distal HisE7 residue as the endogenous ligand. Reversible hexa- to penta-coordination of the heme Fe atom modulates ligand binding properties of Ngb and Cygb. Moreover, Ngb and Cygb display a tunnel/cavity system within the protein matrix held to facilitate ligand channeling to/from the heme, multiple ligand copies storage, multi-ligand reactions, and conformational transitions supporting ligand binding.     

Heme-ligand tunneling in group I truncated hemoglobins

Truncated hemoglobins (trHbs) are small hemoproteins forming a separate cluster within the hemoglobin superfamily; their functional roles in bacteria, plants, and unicellular eukaryotes are marginally understood. Crystallographic investigations have shown that the trHb fold (a two-on-two alpha-helical sandwich related to the globin fold) hosts a protein matrix tunnel system offering a potential path for ligand diffusion to the heme distal site. The tunnel topology is conserved in group I trHbs, although with modulation of its size/structure. Here, we present a crystallographic investigation on trHbs from Mycobacterium tuberculosis, Chlamydomonas eugametos, and Paramecium caudatum, showing that treatment of trHb crystals under xenon pressure leads to binding of xenon atoms at specific (conserved) sites along the protein matrix tunnel. The crystallographic results are in keeping with data from molecular dynamics simulations, where a dioxygen molecule is left free to diffuse within the protein matrix. Modulation of xenon binding over four main sites is related to the structural properties of the tunnel system in the three trHbs and may be connected to their functional roles. In a parallel crystallographic investigation on M. tuberculosis trHbN, we show that butyl isocyanide also binds within the apolar tunnel, in excellent agreement with concepts derived from the xenon binding experiments. These results, together with recent data on atypical CO rebinding kinetics to group I trHbs, underline the potential role of the tunnel system in supporting diffusion, but also accumulation in multiple copies, of low polarity ligands/molecules within group I trHbs.     

Structure-function relationships in the growing hexa-coordinate hemoglobin sub-family

Hemoglobin and related heme proteins, generally referred to as 'globins', reversibly bind gaseous diatomic ligands (O2, NO, and CO) to a penta-coordinate heme iron atom, the ligand filling the sixth coordination site. Over the last decade, several new globins have been reported to display a functionally-relevant hexa-coordinate heme iron atom, whose sixth coordination site is taken by an endogenous protein ligand. The reversible intramolecular hexa- to penta-coordination process at the heme-Fe atom modulates exogenous ligand binding properties of hexa-coordinate globins. Here, we review current knowledge on hexa-coordinate globins in terms of their structural and functional properties.     

Proximal effects in the modulation of nitric oxide synthase reactivity: a QM-MM study

Nitric oxide synthases (NOS) are heme proteins that have a cysteine residue as axial ligand, which generates nitric oxide (NO). The proximal environment, specifically H-bonding between tryptophan (Trp) 178 and thiolate, has been proposed to play a fundamental role in the modulation of NOS activity. We analyzed the molecular basis of this modulation by performing electronic structure calculations on isolated model systems and hybrid quantum-classical computations of the active sites in the protein environment for wild-type and mutant (Trp 178 × Gly) proteins. Our results show that in the ferrous proteins NO exhibits a considerable trans effect. We also showed that in the ferrous (Fe⁺²) mutant NOS the absence of Trp, experimentally associated to a protonated cysteine, weakens the Fe–S bond and yields five coordinate complexes. In the ferric (Fe⁺³) state, the NO dissociation energy is shown to be slightly smaller in the mutant NOS, implying that the Fe⁺³–NO complex has a shorter half-life. We found computational evidence suggesting that ferrous NOS is favored in wild-type NOS when compared to the Trp mutant, consistently with the fact that Trp mutants have been shown to accumulate less Fe⁺²–NO dead end species. We also found that the heme macrocycle showed a significant distortion in the wild-type protein, due to the presence of the nearby Trp 178. This may also play a role in the subtle tuning of the electronic structure of the heme moiety.     

Ultrahigh resolution structures of nitrophorin 4: heme distortion in ferrous CO and NO complexes

Nitrophorin 4 (NP4), a nitric oxide (NO)-transport protein from the blood-sucking insect Rhodnius prolixus, uses a ferric (Fe3+) heme to deliver NO to its victims. NO binding to NP4 induces a large conformational change and complete desolvation of the distal pocket. The heme is markedly nonplanar, displaying a ruffling distortion postulated to contribute to stabilization of the ferric iron. Here, we report the ferrous (Fe2+) complexes of NP4 with NO, CO, and H2O formed after chemical reduction of the protein and the characterization of these complexes by absorption spectroscopy, flash photolysis, and ultrahigh-resolution crystallography (resolutions vary from 0.9 to 1.08 A). The absorption spectra, both in solution and in the crystal, are typical for six-coordinated ferrous complexes. Closure and desolvation of the distal pocket occurs upon binding CO or NO to the iron regardless of the heme oxidation state, confirming that the conformational change is driven by distal ligand polarity. The degree of heme ruffling is coupled to the nature of the ligand and the iron oxidation state in the following order: (Fe3+)-NO > (Fe2+)-NO > (Fe2+)-CO > (Fe3+)-H2O > (Fe2+)-H2O. The ferrous coordination geometry is as expected, except for the proximal histidine bond, which is shorter than typically found in model compounds. These data are consistent with heme ruffling and coordination geometry serving to stabilize the ferric state of the nitrophorins, a requirement for their physiological function. Possible roles for heme distortion and NO bending in heme protein function are discussed.     

Ibuprofen Impairs Allosterically Peroxynitrite Isomerization by Ferric Human Serum Heme-Albumin

Human serum albumin (HSA) participates in heme scavenging; in turn, heme endows HSA with myoglobin-like reactivity and spectroscopic properties. Here, the allosteric effect of ibuprofen on peroxynitrite isomerization to NO₃⁻ catalyzed by ferric human serum heme-albumin (HSA-heme-Fe(III)) is reported. Data were obtained at 22.0 °C. HSA-heme-Fe(III) catalyzes peroxynitrite isomerization in the absence and presence of CO₂; the values of the second order catalytic rate constant (k_on) are 4.1 × 10⁵ and 4.5 × 10⁵ m⁻¹ s⁻¹, respectively. Moreover, HSA-heme-Fe(III) prevents peroxynitrite-mediated nitration of free added l-tyrosine. The pH dependence of k_on (pK_a = 6.9) suggests that peroxynitrous acid reacts preferentially with the heme-Fe(III) atom, in the absence and presence of CO₂. The HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite has been ascribed to the reactive pentacoordinated heme-Fe(III) atom. In the absence and presence of CO₂, ibuprofen impairs dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) and facilitates the nitration of free added l-tyrosine; the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(III) (L) ranges between 7.7 × 10⁻⁴ and 9.7 × 10⁻⁴ m. Under conditions where [ibuprofen] is ≫L, the kinetics of HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite is superimposable to that obtained in the absence of HSA-heme-Fe(III) or in the presence of non-catalytic HSA-heme-Fe(III)-cyanide complex and HSA. Ibuprofen binding impairs allosterically peroxynitrite isomerization by HSA-heme-Fe(III), inducing the hexacoordination of the heme-Fe(III) atom. These results represent the first evidence for peroxynitrite isomerization by HSA-heme-Fe(III), highlighting the allosteric modulation of HSA-heme-Fe(III) reactivity by heterotropic interaction(s), and outlining the role of drugs in modulating HSA functions. The present results could be relevant for the drug-dependent protective role of HSA-heme-Fe(III) in vivo.     

Resonance Raman studies of cytochrome P450BM3 and its complexes with exogenous ligands.

Resonance Raman spectra are reported for both the heme domain and holoenzyme of cytochrome P450BM3 in the resting state and for the ferric NO, ferrous CO, and ferrous NO adducts in the absence and presence of the substrate, palmitate. Comparison of the spectrum of the palmitate-bound form of the heme domain with that of the holoenzyme indicates that the presence of the flavin reductase domain alters the structure of the heme domain in such a way that water accessibility to the distal pocket is greater for the holoenzyme, a result that is consistent with analogous studies of cytochrome P450cam. The data for the exogenous ligand adducts are compared to those previously reported for corresponding derivatives of cytochrome P450cam and document significant and important differences for the two proteins. Specifically, while the binding of substrate induces relatively dramatic changes in the nu(Fe-XY) modes of the ferrous CO, ferric NO, and ferrous NO derivatives of cytochrome P450cam, no significant changes are observed for the corresponding derivatives of cytochrome P450BM3 upon binding of palmitate. In fact, the spectral data for substrate-free cytochrome P450BM3 provide evidence for distortion of the Fe-XY fragment, even in the absence of substrate. This apparent distortion, which is nonexistent in the case of substrate-free cytochrome P450cam, is most reasonably attributed to interaction of the Fe-XY fragment with the F87 phenylalanine side chain. This residue is known to lie very close to the heme iron in the substrate-free derivative of cytochrome P450BM3 and has been suggested to prevent hydroxylation of the terminal, omega, position of long-chain fatty acids.