First observation in the gas phase of the ultrafast electronic relaxation pathways of the S(2) states of heme and hemin

The time evolution of electronically excited heme (iron II protoporphyrin IX, [Fe(II) PP]) and its associated salt hemin (iron III protoporphyrin IX chloride, [Fe(III) PP-Cl]), has been investigated for the first time in the gas phase by femtosecond pump-probe spectroscopy. The porphyrins were excited at 400 nm in the S(2) state (Soret band) and their relaxation dynamics was probed by multiphoton ionization at 800 nm. This time evolution was compared with that of the excited state of zinc protoporphyrin IX [Zn PP] whose S(2) excited state likely decays to the long lived S(1) state through a conical intersection, in less than 100 fs. Instead, for [Fe(II) PP] and [Fe(III) PP-Cl], the key relaxation step from S(2) is interpreted as an ultrafast charge transfer from the porphyrin excited orbital π* to a vacant d orbital on the iron atom (ligand to metal charge transfer, LMCT). This intermediate LMCT state then relaxes to the ground state within 250 fs. Through this work a new, serendipitous, preparation step was found for Fe(II) porphyrins, in the gas phase.     

Design of a five-coordinate heme protein maquette: a spectroscopic model of deoxymyoglobin

The substitution of 1-methyl-l-histidine for the histidine heme ligands in a de novo designed four-alpha-helix bundle scaffold results in conversion of a six-coordinate cytochrome maquette into a self-assembled five-coordinate mono-(1-methyl-histidine)-ligated heme as an initial maquette for the dioxygen carrier protein myoglobin. UV-vis, magnetic circular dichroism, and resonance Raman spectroscopies demonstrate the presence of five-coordinate mono-(1-methyl-histidine) ligated ferrous heme spectroscopically similar to deoxymyoglobin. Thermodynamic analysis of the ferric and ferrous heme dissociation constants indicates greater destabilization of the ferric state than the ferrous state. The ferrous heme protein reacts with carbon monoxide to form a (1-methyl-histidine)-Fe(II)(heme)-CO complex; however, reaction with dioxygen leads to autoxidation and ferric heme dissociation. These results indicate that negative protein design can be used to generate a five-coordinate heme within a maquette scaffold.     

Proton-coupled electron transfer reactions at a heme-propionate in an iron-protoporphyrin-IX model compound

A heme model system has been developed in which the heme-propionate is the only proton donating/accepting site, using protoporphyrin IX-monomethyl esters (PPIX(MME)) and N-methylimidazole (MeIm). Proton-coupled electron transfer (PCET) reactions of these model compounds have been examined in acetonitrile solvent. (PPIX(MME))Fe(III)(MeIm)(2)-propionate (Fe(III)~CO(2)) is readily reduced by the ascorbate derivative 5,6-isopropylidine ascorbate to give (PPIX(MME))Fe(II)(MeIm)(2)-propionic acid (Fe(II)~CO(2)H). An excess of the hydroxylamine TEMPOH or of hydroquinone similarly reduces Fe(III)~CO(2), and TEMPO and benzoquinone oxidize Fe(II)~CO(2)H to return to Fe(III)~CO(2). The measured equilibrium constants, and the determined pK(a) and E(1/2) values, indicate that Fe(II)~CO(2)H has an effective bond dissociation free energy (BDFE) of 67.8 ± 0.6 kcal mol(-1). In these PPIX models, electron transfer occurs at the iron center and proton transfer occurs at the remote heme propionate. According to thermochemical and other arguments, the TEMPOH reaction occurs by concerted proton-electron transfer (CPET), and a similar pathway is indicated for the ascorbate derivative. Based on these results, heme propionates should be considered as potential key components of PCET/CPET active sites in heme proteins.     

Transient weak protein-protein complexes transfer heme across the cell wall of Staphylococcus aureus

Iron is an essential nutrient for the bacterial pathogen Staphylococcus aureus . Heme in hemoglobin (Hb) is the most abundant source of iron in the human body and during infections is captured by S. aureus using iron-regulated surface determinant (Isd) proteins. A central step in this process is the transfer of heme between the cell wall associated IsdA and IsdC hemoproteins. Biochemical evidence indicates that heme is transferred via an activated IsdA:heme:IsdC heme complex. Transfer is rapid and occurs up to 70,000 times faster than indirect mechanisms in which heme is released into the solvent. To gain insight into the mechanism of transfer, we modeled the structure of the complex using NMR paramagnetic relaxation enhancement (PRE) methods. Our results indicate that IsdA and IsdC transfer heme via an ultraweak affinity "handclasp" complex that juxtaposes their respective 3(10) helices and β7/β8 loops. Interestingly, PRE also identified a set of transient complexes that could represent high-energy pre-equilibrium encounter species that form prior to the stereospecific handclasp complex. Targeted amino acid mutagenesis and stopped-flow measurements substantiate the functional relevance of a PRE-derived model, as mutation of interfacial side chains significantly slows the rate of transfer. IsdA and IsdC bind heme using NEAr Transporter (NEAT) domains that are conserved in many species of pathogenic Gram-positive bacteria. Heme transfer in these microbes may also occur through structurally similar transient stereospecific complexes.     

Is δ‐Aminolevulinic Acid Dehydratase Rate Limiting in Heme Biosynthesis Following Exposure of Cells to δ‐Aminolevulinic Acid?¶

Understanding the regulation and control of heme/porphyrin biosynthesis is critical for the optimization of the delta-aminolevulinic-acid (ALA)-mediated photodynamic therapy of cancer, in which endogenously produced protoporphyrin IX (PPIX) is the photosensitizer. The human breast cancer cell line MCF-7, the rat mammary adenocarcinoma cell line R3230AC, the mouse mammary tumor cell line EMT-6 and the human mesothelioma cell line H-MESO-1 were used to study ALA-induced PPIX levels and their relationship to delta-aminolevulinic acid dehydratase (ALA-D) activity in vitro. Incubation of these cell lines with 0.5 mM ALA for 3 h resulted in a significant increase in PPIX accumulation, compared with control cells, but there was no significant change in ALA-D activity. Exposure of cells incubated with ALA to 30 mJ/cm2 of fluorescent light, a dose that would cause a 50% reduction in cell proliferation, did not significantly alter the activity of ALA-D. Increasing the activity of porphobilinogen deaminase (PBGD), the enzyme immediately subsequent to ALA-D, by four- to seven-fold via transfection of cells with PBGD complementary DNA did not alter the activity of ALA-D. However, incubation of cells with various concentrations of succinyl acetone, a potent inhibitor of ALA-D, caused a concomitant decline in both PPIX accumulation and ALA-D activity. These data imply that when cells are exposed to exogenous ALA, ALA-D is an important early-control step in heme/porphyrin biosynthesis and that regulation of PPIX synthesis by this dehydratase may impact the effectiveness of ALA-mediated photosensitization.     

Electron-transfer chemistry of Ru-linker-(heme)-modified myoglobin: rapid intraprotein reduction of a photogenerated porphyrin cation radical

We report the synthesis and characterization of RuC7, a complex in which a heme is covalently attached to a [Ru(bpy)(3)](2+) complex through a -(CH(2))(7)- linker. Insertion of RuC7 into horse heart apomyoglobin gives RuC7Mb, a Ru(heme)-protein conjugate in which [Ru(bpy)(3)](2+) emission is highly quenched. The rate of photoinduced electron transfer (ET) from the resting (Ru(2+)/Fe(3+)) to the transient (Ru(3+)/Fe(2+)) state of RuC7Mb is >10(8) s(-1); the back ET rate (to regenerate Ru(2+)/Fe(3+)) is 1.4 x 10(7) s(-1). Irreversible oxidative quenching by [Co(NH(3))(5)Cl](2+) generates Ru(3+)/Fe(3+): the Ru(3+) complex then oxidizes the porphyrin to a cation radical (P*+); in a subsequent step, P*+ oxidizes both Fe(3+) (to give Fe(IV)=O) and an amino acid residue. The rate of intramolecular reduction of P*+ is 9.8 x 10(3) s(-1); the rate of ferryl formation is 2.9 x 10(3) s(-1). Strong EPR signals attributable to tyrosine and tryptophan radicals were recorded after RuC7MbM(3+) (M = Fe, Mn) was flash-quenched/frozen.     

Dioxygen reactivity of meso -hydroxylated hemes: Intermediates in heme degradation process catalyzed by heme oxygenase

Heme oxygenase (HO) is the only enzyme in mammals known to catalyse the physiological degradation of unwanted heme into biliverdin, Fe ion and CO. The process involves introduction of the hydroxyl group at one of itsmeso-positions as the first fundamental step of the heme cleavage process. It was also found thatmeso-amino heme undergoes similar ring-cleavage process while reacting with dioxygen in presence of pyridine as an axial ligand. The present paper briefly reviews the reactions of modelmeso-hydroxylated heme and its analogues with dioxygen, and their relevance in the heme degradation process.     

Compound I of naked heme (iron protoporphyrin IX)

Gaseous iron protoporphyrin IX (heme) ions, Fe(PP-IX)+, obtained by electrospray ionization of a methanol solution of hemin chloride, are allowed to react with ozone, forming a species that is tentatively assigned the structure of an oxo complex, namely, an oxo iron(IV) protoporphyrin IX radical-cation species, (PP-IX).+FeIV=O. This species, representing the naked core of the putative active oxidant (compound I) of heme enzymes, is characterized by its reactivity behavior in Fourier transform ion cyclotron resonance mass spectrometry, performing as an active O-atom donor. A quite distinct reactivity is displayed by an isomeric species, holding the additional oxygen on the porphyrin frame, Fe(PP-IX(O))+. This isomer undergoes a ligand addition process, as was previously observed for Fe(PP-IX)+.     

Base-promoted acyloin rearrangement of 1,8-di-tert-butyldimethylsilyloxybicyclo[2.2.2]oct-5-en-2-ones

Treatment of 1,8-di-tert-butyldimethylsilyloxybicyclo[2.2.2]oct-5-en-2-ones having an electron-withdrawing group such as a nitro, formyl, cyano, and imido group at C-7 with a strong base (potassium hydride, or potassium bistrimethylsilylamide, etc.), resulted in an acyloin rearrangement reaction accompanied by retention of two silyloxy groups to afford 1,8-disilyloxybicyclo[3.2.1]oct-3-en-2-ones.     

Electroactive core-shell nanocluster films of heme proteins, polyelectrolytes, and silica nanoparticles

Novel protein core-shell nanocluster films were assembled layer by layer on solid surfaces. In the first step, positively charged heme protein hemoglobin (Hb) or myoglobin (Mb) and negatively charged poly(styrenesulfonate) (PSS) were alternately adsorbed on the surface of SiO2 nanoparticles, forming core-shell SiO2-(protein/PSS)m nanoclusters. In the second step, the SiO2-(protein/PSS)m nanoclusters and polycationic poly(ethylenimine) (PEI) were assembled layer by layer on various solid substrates, forming [[SiO2-(protein/PSS)m]/PEI]n films. Various techniques were used to characterize the nanoclusters and monitor the film growth. [[SiO2-(protein/PSS)m]/PEI]n films at pyrolytic graphite (PG) electrodes exhibited well-defined, chemically reversible cyclic voltammetric reduction-oxidation peaks characteristic of the heme Fe(III)/Fe(II) redox couples. The proteins in the films retained near native conformations in the medium pH range, and the films catalyzed electrochemical reduction of oxygen and hydrogen peroxide. Advantages of the nanocluster films over the simple [SiO2/protein]n layer-by-layer films include a larger fraction of electroactive protein and higher specific biocatalytic activity. Using this approach, biocatalytic activity can be tailored and controlled by varying the number of bilayers deposited on the nanoparticle cores and the number of nanocluster layers on electrodes.     

Heme protein oxygen affinity regulation exerted by proximal effects

Heme proteins are found in all living organisms and are capable of performing a wide variety of tasks, requiring in many cases the binding of diatomic ligands, namely, O(2), CO, and/or NO. Therefore, subtle regulation of these diatomic ligands' affinity is one of the key issues for determining a heme protein's function. This regulation is achieved through direct H-bond interactions between the bound ligand and the protein, and by subtle tuning of the intrinsic heme group reactivity. In this work, we present an investigation of the proximal regulation of oxygen affinity in Fe(II) histidine coordinated heme proteins by means of computer simulation. Density functional theory calculations on heme model systems are used to analyze three proximal effects: charge donation, rotational position, and distance to the heme porphyrin plane of the proximal histidine. In addition, hybrid quantum-classical (QM-MM) calculations were performed in two representative proteins: myoglobin and leghemoglobin. Our results show that all three effects are capable of tuning the Fe-O(2) bond strength in a cooperative way, consistently with the experimental data on oxygen affinity. The proximal effects described herein could operate in a large variety of O(2)-binding heme proteins-in combination with distal effects-and are essential to understand the factors determining a heme protein's O(2) affinity.     

Role of heme types in heme-copper oxidases: effects of replacing a heme b with a heme o mimic in an engineered heme-copper center in myoglobin

To address the role of the secondary hydroxyl group of heme a/o in heme-copper oxidases, we incorporated Fe(III)-2,4 (4,2) hydroxyethyl vinyl deuterioporphyrin IX, as a heme o mimic, into the engineered heme-copper center in myoglobin (sperm whale myoglobin L29H/F43H, called Cu(B)Mb). The only difference between the heme b of myoglobin and the heme o mimic is the substitution of one of the vinyl side chains of the former with a hydroxyethyl group of the latter. This substitution resulted in an approximately 4 nm blue shift in the Soret band and approximately 20 mV decrease in the heme reduction potential. In a control experiment, the heme b in Cu(B)Mb was also replaced with a mesoheme, which resulted in an approximately 13 nm blue shift and approximately 30 mV decrease in the heme reduction potential. Kinetic studies of the heme o mimic-substituted Cu(B)Mb showed significantly different reactivity toward copper-dependent oxygen reduction from that of the b-type Cu(B)Mb. In reaction with O2, Cu(B)Mb with a native heme b showed heme oxygenase activity by generating verdoheme in the presence of Cu(I). This heme degradation reaction was slowed by approximately 19-fold in the heme o mimic-substituted Cu(B)Mb (from 0.028 s(-1) to 0.0015 s(-1)), while the mesoheme-substituted Cu(B)Mb shared a similar heme degradation rate with that of Cu(B)Mb (0.023 s(-1)). No correlation was found between the heme reduction potential and its O2 reactivity. These results strongly suggest the critical role of the hydroxyl group of heme o in modulating heme-copper oxidase activity through participation in an extra hydrogen-bonding network.     

Synthesis of 1-hydroxy-substituted pyrazolo[3,4-c]- and pyrazolo[4, 3-c]quinolines and -isoquinolines from 4- and 5-aryl-substituted 1-benzyloxypyrazoles

1-Hydroxypyrazolo[3,4-c]quinoline (22), 1-hydroxypyrazolo[4, 3-c]quinoline (21), 1-hydroxypyrazolo[3,4-c]isoquinoline (20), and 1-hydroxypyrazolo[4,3-c]isoquinoline (19) were prepared from 1-benzyloxypyrazole (6), establishing the pyridine B-ring in the terminal step. The pyridine ring of pyrazoloquinolines 14 and 18 was formed via cyclization of a formyl group at C-4 or C-5 and an amino group of a 2-aminophenyl substituent at C-5 or C-4 in 1-benzyloxypyrazole. The pyridine ring of pyrazoloisoquinolines 5 and 9 was created via cyclization of a formyl group in a 2-formylphenyl substituent at C-4 or C-5 with an iminophosphorane group installed at C-5 or C-4 of 1-benzyloxypyrazole by lithiation followed by reaction with tosyl azide and then with tributylphoshine utilizing the Staudinger/aza-Wittig protocol. The 2-aminophenyl and the 2-formylphenyl substituent were introduced at C-5 or C-4 by regioselective metalation followed by transmetalation to the pyrazolylzinc halide and subsequent palladium-catalyzed cross-coupling with 2-iodoaniline or 2-bromobenzaldehyde. The order of reactions and use of protecting groups in the individual sequences have been optimized. The 1-benzyloxy-substituted pyrazoloquinolines and isoquinolines thus obtained were debenzylated by strong acid to the corresponding 1-hydroxy-substituted pyrazoloquinolines and isoquinolines 19-22.     

Self-assembly of highly ordered Peptide amphiphile metalloporphyrin arrays

Long fibers assembled from peptide amphiphiles capable of binding the metalloporphyrin zinc protoporphyrin IX ((PPIX)Zn) have been synthesized. Rational peptide design was employed to generate a peptide, c16-AHL(3)K(3)-CO(2)H, capable of forming a β-sheet structure that propagates into larger fibrous structures. A porphyrin-binding site, a single histidine, was engineered into the peptide sequence in order to bind (PPIX)Zn to provide photophysical functionality. The resulting system indicates control from the molecular level to the macromolecular level with a high order of porphyrin organization. UV/visible and circular dichroism spectroscopies were employed to detail molecular organization, whereas electron microscopy and atomic force microscopy aided in macromolecular characterization. Preliminary picosecond transient absorption data are also reported. Reduced hemin, (PPIX)Fe(II), was also employed to highlight the material's versatility and tunability.     

Chiral recognition of zinc(II) ion complexes composed of bicyclo[3.3.0] octane-2,6-diol and <Emphasis Type="Italic">s</Emphasis>-Naproxen probed by collisional-induced dissociation

Chiral recognition of racemic bicyclo[3.3.0] octane-2,6-diol(B) was achieved in the gas phase using s-Naproxen(A) as reference, using the kinetics of competitive unimolecule dissociation of tetrameric zinc(II)-bound complexes by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer(ESI-FTMS). As undergoing a mild competitive collision-induced dissociation(CID) experiment with a constant pressure argon gas introduced by leak valve, the tetrameric cluster ion [A(2)B(2)Z(n)(II)-H](+) forms only two trimeric ions and R(chiral) is subsequently obtained in the kinetic method. Further studies obtained the difference of Gibbs free energy of [ABZ(n)(II)-H](+)(Delta Delta G(ABZn(II)-H](+))) by dissociating [A(2)BZ(n)(II)-H](+), resulting two fragment ions [ABZ(n)(II)-H](+) and [A(2)Z(n)(II)-H](+), which can be established to a linear relationship between Delta Delta G([ABZn(II)-H](+)) and R(chiral)' basing on the kinetic method. The value of R(chiral)' suggested that Delta Delta G([ABZn(II)-H](+)) could be regarded as zero. Meanwhile, dissociation of [AB(2)Z(n)(II)-H](+) generated only one daughter ion [ABZ(n)(II)-H](+) in a stable pressure. Thus, a linear relationship was established between the difference of Gibbs free energy of [AB(2)Z(n)(II)-H](+)(Delta Delta G([AB(2)Zn(II)-H](+))) and R(chiral)" if the Delta Delta G([ABZn(II)-H](+)) can be negligible. Because there is also a linear relationship of R(chiral) in the tetrameric ion [A(2)B(2)Z(n)(II)-H](+) and the Gibbs energy difference of trimeric cluster ion [A(2)BZ(n)(+)(II)-H](Delta Delta G([A(2)BZn(II)-H](+))) plus that of [AB(2)Z(n)(II)-H](+), Delta Delta G([A(2)BZ(n)(II)-H]+]) is easy to be calculated in the dissociation process of tetrameric ion. Stable of R(chiral), R(chiral)' and R(chiral)" under different pressures show T(eff) does not affect the chiral recognition of cluster ions in the condition selected. If an only-one-daughter-ion fragment process of [A(2)BZ(n)(II)-H](+) was existed, R(chiral)' relating to this dissociation would be calculated just like R(chiral)" of [AB(2)Z(n)(II)-H](+) does. Conclusion was obtained that [A(2)BZ(n)(II)-H](+) makes more contribution to chiral recognition of tetrameric ion measured by kinetic method than [AB(2)Z(n)(II)-H](+) does as R(chiral)' and R(chiral)" were applied as index to evaluate the Gibbs free energy difference of these two trimeric cluster ions. Further discussion shows that steric interactions and pi-pi stacking interactions are the major factors responsible for the observed efficient chiral recognition in this system.     

Genetic engineering of the heme pocket in human serum albumin: modulation of O2 binding of iron protoporphyrin IX by variation of distal amino acids

Complexing an iron protoporphyrin IX into a genetically engineered heme pocket of recombinant human serum albumin (rHSA) generates an artificial hemoprotein, which can bind O2 in much the same way as hemoglobin (Hb). We previously demonstrated a pair of mutations that are required to enable the prosthetic heme group to bind O2 reversibly: (i) Ile-142-->His, which is axially coordinated to the central Fe2+ ion of the heme, and (ii) Tyr-161-->Phe or Leu, which makes the sixth coordinate position available for ligand interactions [I142H/Y161F (HF) or I142H/Y161L (HL)]. Here we describe additional new mutations designed to manipulate the architecture of the heme pocket in rHSA-heme complexes by specifically altering distal amino acids. We show that introduction of a third mutation on the distal side of the heme (at position Leu-185, Leu-182, or Arg-186) can modulate the O2 binding equilibrium. The coordination structures and ligand (O2 and CO) binding properties of nine rHSA(triple mutant)-heme complexes have been physicochemically and kinetically characterized. Several substitutions were severely detrimental to O2 binding: for example, Gln-185, His-185, and His-182 all generated a weak six-coordinate heme, while the rHSA(HF/R186H)-heme complex possessed a typical bis-histidyl hemochrome that was immediately autoxidized by O2. In marked contrast, HSA(HL/L185N)-heme showed very high O2 binding affinity (P1/2O2 1 Torr, 22 degrees C), which is 18-fold greater than that of the original double mutant rHSA(HL)-heme and very close to the affinities exhibited by myoglobin and the high-affinity form of Hb. Introduction of Asn at position 185 enhances O2 binding primarily by reducing the O2 dissociation rate constant. Replacement of polar Arg-186 with Leu or Phe increased the hydrophobicity of the distal environment, yielded a complex with reduced O2 binding affinity (P1/2O2 9-10 Torr, 22 degrees C), which nevertheless is almost the same as that of human red blood cells and therefore better tuned to a role in O2 transport.     

Quantum mechanical/molecular mechanical study of mechanisms of heme degradation by the enzyme heme oxygenase: the strategic function of the water cluster

Heme degradation by heme oxygenase (HO) enzymes is important in maintaining iron homeostasis and prevention of oxidative stress, etc. In response to mechanistic uncertainties, we performed quantum mechanical/molecular mechanical investigations of the heme hydroxylation by HO, in the native route and with the oxygen surrogate donor H2O2. It is demonstrated that H2O2 cannot be deprotonated to yield Fe(III)OOH, and hence the surrogate reaction starts from the FeHOOH complex. The calculations show that, when starting from either Fe(III)OOH or Fe(III)HOOH, the fully concerted mechanism involving O-O bond breakage and O-C(meso) bond formation is highly disfavored. The low-energy mechanism involves a nonsynchronous, effectively concerted pathway, in which the active species undergoes first O-O bond homolysis followed by a barrier-free (small with Fe(III)HOOH) hydroxyl radical attack on the meso position of the porphyrin. During the reaction of Fe(III)HOOH, formation of the Por+*FeIV=O species, compound I, competes with heme hydroxylation, thereby reducing the efficiency of the surrogate route. All these conclusions are in accord with experimental findings (Chu, G. C.; Katakura, K.; Zhang, X.; Yoshida, T.; Ikeda-Saito, M. J. Biol. Chem. 1999, 274, 21319). The study highlights the role of the water cluster in the distal pocket in creating "function" for the enzyme; this cluster affects the O-O cleavage and the O-Cmeso formation, but more so it is responsible for the orientation of the hydroxyl radical and for the observed alpha-meso regioselectivity of hydroxylation (Ortiz de Montellano, P. R. Acc. Chem. Res. 1998, 31, 543). Differences/similarities with P450 and HRP are discussed.     

Coulomb effects in binding of heme in gas-phase ions of myoglobin

Coulomb effects in binding of heme in gas-phase holomyoglobin ions are studied. Positive and negative ions are formed from solution myoglobin with Fe(2+) (ferromyoglobin) and Fe(3+) (ferrimyoglobin). The energy that must be added to the resulting holomyoglobin ions to cause heme loss has been measured by triple-quadrupole tandem mass spectrometry. With negative ions, neutral heme is lost regardless of the charge state of Fe in solution. It is likely that the Fe(3+) is reduced to Fe(2+) in the negative electrospray process. With positive ions, predominantly neutral heme loss is observed with ions formed from ferromyoglobin in solution, and positive heme loss with ions formed from ferrimyoglobin in solution. The energies required to induce neutral heme loss are similar for positive and negative ions. The energies required to induce charged heme loss from positive holomyoglobin ions are significantly less. Coulomb repulsion between the charged heme and charged protein appears to lower the barrier for heme loss. These results are consistent with a simple model potential with a long-range Coulomb repulsion and short-range attraction between the heme and protein.     

Synthesis,characterization, and laser flash photolysis reactivity of a carbonmonoxy heme complex

We present here the synthesis, characterization, and flash photolysis study of [(F(8)TPP)Fe(II)(CO)(THF)] (1) [F(8)TPP = tetrakis(2,6-difluorophenyl)porphyrinate(2-)]. Complex 1 crystallizes from THF/heptane solvent system as a tris-THF solvate, [(F(8)TPP)Fe(II)(CO)(THF)].3THF (1.3THF), with ferrous ion in the porphyrin plane (C(61)H(52)F(8)FeN(4)O(5); a = 11.7908(2) A, b = 20.4453(2) A, c = 39.9423(3), alpha = 90 degrees, beta = 90 degrees, gamma = 90 degrees; orthorhombic, P2(1)2(1)2(1), Z = 8; Fe-N(4)(av) = 2.00 A; N-Fe-N (all) = 90.0 degrees ). This complex (as 1.THF) has also been characterized by (1)H NMR [six-coordinate, low-spin heme; CD(3)CN, RT, delta 8.82 (s, pyrrole-H, 8H), 7.89 (s, para-phenyl-H, 8H), 7.46 (s, meta-phenyl-H, 4H), 3.58 (s, THF, 8H), 1.73 (s, THF, 8H)], (2)H NMR (pyrrole-deuterated analogue) [(F(8)TPP-d(8))Fe(II)(CO)(THF)] [THF, RT, delta 8.78 ppm (s, pyrrole-D)], (13)C NMR (on (13)CO-enriched adduct) [THF-d(8), RT, delta 206.5 ppm; CD(2)Cl(2), RT, delta 206.1 ppm], UV-vis [THF, RT, lambda(max), 411 (Soret), 525 nm], and IR [293 K, solution, nu(CO) 1979 cm(-)(1) (THF), 1976 cm(-)(1) (acetone), 1982 cm(-)(1) (CH(3)CN)] spectroscopies. In order to more fully understand the intricacies of solvent-ligand binding (as compared to CO rebinding to the photolyzed heme), we have also synthesized the bis-THF adduct [(F(8)TPP)Fe(II)(THF)(2)]. Complex 2 also crystallizes from THF/heptane solvent system as a bis-THF solvate, [(F(8)TPP)Fe(II)(THF)(2)].2THF (2.2THF), with ferrous iron in the porphyrin plane (C(60)H(52)F(8)FeN(4)O(4); a = 21.3216(3) A, b = 12.1191(2) A, c = 21.0125(2) A, alpha = 90 degrees, beta = 105.3658(5) degrees, gamma = 90 degrees; monoclinic, C2/c, Z = 4; Fe-N(4)(av) = 2.07 A; N-Fe-N (all) = 90.0 degrees ). Further characterization of 2 includes UV-vis [THF, lambda(max), 421 (Soret), 542 nm] and (1)H NMR [six-coordinate, high spin heme; THF-d(8), RT, delta 56.7 (s, pyrrole-H, 8H), 8.38 (s, para-phenyl-H, 8H), 7.15 (s, meta-phenyl-H, 4H)] spectroscopies. Flash photolysis studies employing 1 were able to resolve the CO rebinding kinetics in both THF and cyclohexane solvents. In CO saturated THF [[CO] approximately 5 mM] and at [1] congruent with 5 microM, the conversion of [(F(8)TPP)Fe(II)(THF)(2)] (produced after photolytic displacement of CO) to [(F(8)TPP)Fe(II)(CO)(THF)] was monoexponential, with k(obs) = 1.6 (+/-0.2) x 10(4) s(-)(1). Reduction in [CO] by vigorous Ar purging gave k(obs) congruent with 10(3) s(-)(1) in cyclohexane. The study presented in this report lays the foundation for applying fast-time scale studies based on CO flash photolysis to the more complicated heterobimetallic heme/Cu systems.