Sixth Ligand Research Articles

Substrate binding triggers a switch in the iron coordination in dehaloperoxidase from Amphitrite Ornate

We have explored the effect of substrate binding on the heme iron conformation in the enzyme dehaloperoxidase (DHP). DHP is a dimeric hemoglobin that also has significant peroxidase activity under physiological conditions and has been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that uses hydrogen peroxide as a cosubstrate. Hyperfine sublevel correlation spectroscopic (HYSCORE) analysis of the ferric form of DHP was carried out to characterize effects of the substrate 2,4,6-trifluorophenol (TFP) binding on the iron coordination in order to elucidate molecular mechanisms responsible for switching the protein function from a globin to a peroxidase. The CW EPR spectrum shows that at pH 6.0 DHP heme iron exists in a highly axial high spin (HS) state that could be interpreted as arising from two different populations of the HS iron centers. Substrate binding does not change the spin state at pH 6.0, however, affects the magnetic parameters of the signal. HYSCORE spectra recorded at magnetic field corresponding to g = 2 revealed the presence of exchangeable protons with hyperfine coupling of ca. 6 MHz, consistent with a water molecule being the sixth ligand in the iron coordination. These protons' spectral features disappeared upon substrate binding. At pH 9.6 the EPR spectrum from heme iron of DHP shows the presence of both high- and low-spin states with the low spin signal characteristic of hydroxyl form. Upon TFP binding the low spin signal disappears. HYSCORE spectra at pH 9.6 also show the presence of exchangeable protons that disappear upon substrate binding. This observation highlights the proposed role of molecules in the distal pocket to control the peroxidase function of DHP.Supported by the NSF Grant MCB-0451510 to T.I.S. and ARO grant 52278-LS to S.F.

Insights Into The Mechanism Of The Cobalt Containing Nitrile Hydratase From Geobacillus Pallidus

The crystal structures of the wild-type, Co(III) containing, nitrile hydratase and the mutant (βF36L / βL103S / βY127N / αD4G) enzyme from have been solved at resolutions of 1.4A and 1.15A respectively. Nitrile hydratases are noted for having an unusual cysteine claw structure at the active site. An important observation made in this paper is that cysteines α119 and α121 were both modified to cysteine sulfinic acid instead of the α121 cysteine sulfenic and α119 cysteine sulfinic acid reported previously. This was confirmed by MALDI-TOF mass spectroscopy.The nitrile hydratases catalyse the conversion of nitriles to the corresponding amides. These enzymes underlie the industrial production ofacrylamide. In the cysteine claw structure an Fe(III) or Co(III) ion is octahedrally co-ordinated to three cysteines, two of which are oxidized, and two peptide backbone amide groups. The sixth ligand, trans to the unmodified cysteine is a water molecule or a hydroxide ion. The cysteine claw has the sequence CTLCSC in the iron case and CSLCSC in the cobalt case. The enzyme itself is a α2β2 tetramer with the cysteine claw located in the α subunit and much of the active site contributed by the β subunit.Our crystal structures are almost certainly an inactive form of the enzyme, being crystallized in 100 mM MES (2[N-Morpholino]ethanesulfonic acid) which was found to be an inhibitor of the enzyme subsequent to the crystallization. However at present no evidence suggests that the inactivation is due to the oxidation of the active site cysteine sulfenic acid.The structures give insight into the determinants of the specificity of the enzyme, suggesting that βphe55 and βphe55 obstruct access to the cysteine claw complex for larger substrates allowing the hydration of only smaller aliphatic nitriles.

Structural Probes Of Reactive Intermediates Of Dehaloperoxidase From Amphitrite ornata

The enzyme dehaloperoxidase (DHP) from the marine worm Amphitrite ornata is a unique hemoglobin that functions as a peroxidase, capable of converting 2,4,6-trihalo- phenols (TBP, TCP, and TFP) into the corresponding 2,6-dihalogenated quinones as well as other products. In this overview talk we discuss the structure and function of DHP using X-ray crystallography and nuclear magnetic resonance (NMR) to discuss the large differences between DHP function and hemoglobin function despite the strong structural similarities. The position of halogenated phenols inside the distal pocket is one anomalous feature of DHP that is not observed any other hemoglobin or myoglobin. The X-ray crystal structure of DHP reveals that the distal histidine is flexible and has two major conformations. The closed conformation (named by analogy with Sperm Whale myoglobin) is enforced by the binding of a sixth ligand to the heme iron. In the open conformation, observed in the deoxy DHP X-ray crystal structure, the distal histidine is in a solvent exposed conformation. The role of the histidine in coupling the binding of substrate and inhibitors will be discussed in the context of the mechanism for formation of compound I, compound II and a novel intermediate called compound RH that appears to be crucial to the cycling of DHP between hemoglobin and peroxidase function. The NMR data reveal that there are both interior and exterior binding sites for the substrate. This aspect will be discussed along with evidence from optical and EPR spectroscopy to understand the electron transfer kinetics of DHP.

Dynamics And Energetics Associated With Ligand Photodissociation From Co Bound Chloroperoxidase

Chloroperoxidase (CPO) is the most versatile heme peroxidase that can function as peroxidase, catalase, halogenase/dehalogenase and monooxigenase. The active site of CPO strongly resembles that of cytochrome P450 since the heme iron in both proteins is coordinated with a Cys axial ligand. Two hydrophobic channels that connect the heme binding cavity with the surrounding are likely to provide ligand access into the active site. At alkaline pH the protein undergoes a conformational transition resulting in the population of six coordinated heme iron with the histidine residue in the position of the sixth ligand. Here we report the results of the photoacoustic study of ligand dissociation from CPO. The ligand photodissociation and migration out of the protein matrix occurs within 50 ns and no additional kinetics was determined (detected?) between 50 ns and 5 μs. At acidic pH ligand dissociation is associated with a positive volume change (ΦΔV = 10.9 ± 0.9 mL mol−1) and enthalpy change (ΦΔH = 24 ± 5 kcal mol−1), strongly indicating that the ligand release from the protein matrix does not result in significant changes in protein conformation and CO is likely to migrate through one of the hydrophobic channels. On the other hand, the thermodynamic parameters associated with CO photorelease at pH 10 are significantly smaller than those determined at acidic pH (ΦΔV = 3.2 ± 1.1 mL mol−1 and ΦΔH = -12.1 ± 5.3 kcal mol−1) due to changes in heme coordination upon CO dissociation.

Nitration of Solvent-exposed Tyrosine 74 on Cytochrome c Triggers Heme Iron-Methionine 80 Bond Disruption: NUCLEAR MAGNETIC RESONANCE AND OPTICAL SPECTROSCOPY STUDIES

Cytochrome c, a mitochondrial electron transfer protein containing a hexacoordinated heme, is involved in other physiologically relevant events, such as the triggering of apoptosis, and the activation of a peroxidatic activity. The latter occurs secondary to interactions with cardiolipin and/or post-translational modifications, including tyrosine nitration by peroxynitrite and other nitric oxide-derived oxidants. The gain of peroxidatic activity in nitrated cytochrome c has been related to a heme site transition in the physiological pH region, which normally occurs at alkaline pH in the native protein. Herein, we report a spectroscopic characterization of two nitrated variants of horse heart cytochrome c by using optical spectroscopy studies and NMR. Highly pure nitrated cytochrome c species modified at solvent-exposed Tyr-74 or Tyr-97 were generated after treatment with a flux of peroxynitrite, separated, purified by preparative high pressure liquid chromatography, and characterized by mass spectrometry-based peptide mapping. It is shown that nitration of Tyr-74 elicits an early alkaline transition with a pKa = 7.2, resulting in the displacement of the sixth and axial iron ligand Met-80 and replacement by a weaker Lys ligand to yield an alternative low spin conformation. Based on the study of site-specific Tyr to Phe mutants in the four conserved Tyr residues, we also show that this transition is not due to deprotonation of nitro-Tyr-74, but instead we propose a destabilizing steric effect of the nitro group in the mobile Omega-loop of cytochrome c, which is transmitted to the iron center via the nearby Tyr-67. The key role of Tyr-67 in promoting the transition through interactions with Met-80 was further substantiated in the Y67F mutant. These results therefore provide new insights into how a remote post-translational modification in cytochrome c such as tyrosine nitration triggers profound structural changes in the heme ligation and microenvironment and impacts in protein function.

Cobalt(III) complexes of the type cis-β-[Co(trien)(4-R-Py)CI]CI2 : Synthesis, spectral characterization and redox behaviour

Six new cobalt(III) complexes of the type cis-β-[Co(trien)(4-R-Py)Cl]C 2 , where R = H, Me, Et, t-Bu, COMe and CN have been prepared and characterized by elemental analysis, UV-Vis, FT-IR and 1 H NMR spectral techniques. The results of the spectral studies revealed that these complexes posses a cis-β arrangement of ligands around the metal ion. Molar conductance measurements supported the 1: 2 nature of these complexes. Magnetic susceptibility measurements indicated that the complexes were diamagnetic. Thermal studies revealed that these complexes are thermally stable up to 115 °C. The redox potential values obtained from differential pulse voltammetric technique of these complexes indicated that the redox behaviour depends on the nature of the sixth ligand.

Novel haem co-ordination variants of flavocytochrome P450 BM3

Bacillus megaterium flavocytochrome P450 BM3 is a catalytically self-sufficient fatty acid hydroxylase formed by fusion of soluble NADPH–cytochrome P450 reductase and P450 domains. Selected mutations at residue 264 in the haem (P450) domain of the enzyme lead to novel amino acid sixth (distal) co-ordination ligands to the haem iron. The catalytic, spectroscopic and thermodynamic properties of the A264M, A264Q and A264C variants were determined in both the intact flavocytochromes and haem domains of P450 BM3. Crystal structures of the mutant haem domains demonstrate axial ligation of P450 haem iron by methionine and glutamine ligands trans to the cysteine thiolate, creating novel haem iron ligand sets in the A264M/Q variants. In contrast, the crystal structure of the A264C variant reveals no direct interaction between the introduced cysteine side chain and the haem, although EPR data indicate Cys264 interactions with haem iron in solution. The A264M haem potential is elevated by comparison with wild-type haem domain, and substrate binding to the A264Q haem domain results in a ∼360 mV increase in potential. All mutant haem domains occupy the conformation adopted by the substrate-bound form of wild-type BM3, despite the absence of added substrate. The A264M mutant (which has higher dodecanoate affinity than wild-type BM3) co-purifies with a structurally resolved lipid. These data demonstrate that a single mutation at Ala264 is enough to perturb the conformational equilibrium between substrate-free and substrate-bound P450 BM3, and provide firm structural and spectroscopic data for novel haem iron ligand sets unprecedented in Nature.

Square-pyramidal iron coordination modules as potential spin switches for the chemisorption on gold

Octahedral iron(II) complexes of a unique pyridine-derived tetrapodal pentadentate polyamine ligand, 2,6-C5H3N(CMe[CH2NH2]2)2, show temperature-dependent spin crossover (SCO) depending on the nature of a sixth monodentate ligand L (imidazol or pyridine derivative). For L = 1-methylimidazol, the redox behaviour of the complex, as determined by cyclic voltammetry, suggests an accompanying ligand exchange. Pyridine-4-thiol and the disulphides: 4-(2-methyldisulphanyl)pyridine, 4-(2-hexadecyldisulphanyl)pyridine and 1,2-bis([pyridine-4-yl]methyl)disulphane, were studied as mono-dentate ligands L, with a view to enable chemisorption of iron(II) complexes on a gold surface. In the case of pyridine-4-thiol, the participation of the thiolate functional group in iron coordination is difficult to suppress, whereas the disulphides enter into yet unrecorded redox chemistry with iron(II), yielding a di-iron(III) complex containing a persulphide bridge (S22−).

Ruffling of Metalloporphyrins Bound to IsdG and IsdI, Two Heme-degrading Enzymes in Staphylococcus aureus

IsdG and IsdI are paralogous proteins that are intracellular components of a complex heme uptake system in Staphylococcus aureus. IsdG and IsdI were shown previously to reductively degrade hemin. Crystal structures of the apoproteins show that these proteins belong to a newly identified heme degradation family distinct from canonical eukaryotic and prokaryotic heme oxygenases. Here we report the crystal structures of an inactive N7A variant of IsdG in complex with Fe(3+)-protoporphyrin IX (IsdG-hemin) and of IsdI in complex with cobalt protoporphyrin IX (IsdI-CoPPIX) to 1.8 A or better resolution. These structures show that the metalloporphyrins are buried into similar deep clefts such that the propionic acids form salt bridges to two Arg residues. His(77) (IsdG) or His(76) (IsdI), a critical residue required for activity, is coordinated to the Fe(3+) or Co(3+) atoms, respectively. The bound porphyrin rings form extensive steric interactions in the binding cleft such that the rings are highly distorted from the plane. This distortion is best described as ruffled and places the beta- and delta-meso carbons proximal to the distal oxygen-binding site. In the IsdG-hemin structure, Fe(3+) is pentacoordinate, and the distal side is occluded by the side chain of Ile(55). However, in the structure of IsdI-CoPPIX, the distal side of the CoPPIX accommodates a chloride ion in a cavity formed through a conformational change in Ile(55). The chloride ion participates in a hydrogen bond to the side chain amide of Asn(6). Together the structures suggest a reaction mechanism in which a reactive peroxide intermediate proceeds with nucleophilic oxidation at the beta- or delta-meso carbon of the hemin.

The role of the heme distal ligand in the post-translational modification of Synechocystis hemoglobin

Synechocystis sp. PCC 6803 hemoglobin is a cyanobacterial Group I truncated hemoglobin. In the absence of an exogenous ligand, its single heme group is coordinated by His46 (E10, distal) and His70 (F8, proximal). The protein can undergo a post-translational modification by which His117 (H16, in the C-terminal helix) reacts with the heme 2-vinyl group to form a Markownikoff adduct. The new C–N bond prevents heme loss, alters the dynamics of the protein, and influences ligand binding to the heme group. To explore the factors conditioning the formation of the cross-link, variants of the protein that contained an alanine or a leucine at position 46 (E10) were prepared. A double replacement (His46Leu and Tyr22 (B10) to Phe) was also performed to perturb the network of interactions stabilizing bound exogenous ligand. The single and double replacements affected the optical and NMR properties of the globin, each in a different fashion. Heme–protein cross-linking, as promoted by sodium dithionite, was retarded by the replacement of His46, but reactivity was recovered when imidazole or cyanide was used as exogenous ligand. In addition, a significant amount of a second product was systematically obtained when dithionite treatment was performed on the cyanide-bound proteins. This species was identified by NMR spectroscopy to be an adduct to the 4-vinyl group. It was concluded that the specificity and rate of the cross-linking reaction depended critically on the nature of the sixth ligand to the heme iron.

Syntheses, X-ray Structures, Solid State High-Field Electron Paramagnetic Resonance, and Density-Functional Theory Investigations on Chloro and Aqua MnII Mononuclear Complexes with Amino-Pyridine Pentadentate Ligands

The two pentadentate amino-pyridine ligands L5(2) and L5(3) (L5(2) and L5(3) stand for the N-methyl-N,N',N'-tris(2-pyridylmethyl)ethane-1,2-diamine and the N-methyl-N,N',N'-tris(2-pyridylmethyl)propane-1,3-diamine, respectively) were used to synthesize four mononuclear Mn(II) complexes, namely [(L5(2))MnCl](PF6) (1(PF6)), [(L5(3))MnCl](PF6) (2(PF6)), [(L5(2))Mn(OH2)](BPh4)2 (3(BPh4)2), and [(L5(3))Mn(OH2)](BPh4)2 (4(BPh4)2). The X-ray diffraction studies revealed different configurations for the ligand L5(n) (n = 2, 3) depending on the sixth exogenous ligand and/or the counterion. Solid state high-field electron paramagnetic resonance spectra were recorded on complexes 1-4 as on previously described mononuclear Mn(II) systems with tetra- or hexadentate amino-pyridine ligands. Positive and negative axial zero-field splitting (ZFS) parameters D were determined whose absolute values ranged from 0.090 to 0.180 cm(-1). Density-functional theory calculations were performed unraveling that, in contrast with chloro systems, the spin-spin and spin-orbit coupling contributions to the D-parameter are comparable for mixed N,O-coordination sphere complexes.

Coordination Polymers Based on Aluminum(III) Porphyrins

Aluminum(III) porphyrin carboxylate complexes have shown an affinity for a sixth nitrogenous ligand. The use of isonicotinic or nicotinic acid, which offers both a carboxylate and a nitrogen donor in the same molecule, resulted in the formation of one-dimensional (1-D) coordination polymers. The complexes and their linear oligomers have been characterized by (1)H NMR spectroscopy and nanoelectrospray ionization spectrometry. X-ray analyses confirmed the formation of the 1-D polymers in the solid state.

Strategic Roles of Axial Histidines in Structure Formation and Redox Regulation of Tetraheme Cytochrome c3

Tetraheme cytochrome c 3 (cyt c 3) exhibits extremely low reduction potentials and unique properties. Since axial ligands should be the most important factors for this protein, every axial histidine of Desulfovibrio vulgaris Miyazaki F cyt c 3 was replaced with methionine, one by one. On mutation at the fifth ligand, the relevant heme could not be linked to the polypeptide, revealing the essential role of the fifth histidine in heme linking. The fifth histidine is the key residue in the structure formation and redox regulation of a c-type cytochrome. A crystal structure has been obtained for only H25M cyt c 3. The overall structure was not affected by the mutation except for the sixth methionine coordination at heme 3. NMR spectra revealed that each mutated methionine is coordinated to the sixth site of the relevant heme in the reduced state, while ligand conversion takes place at hemes 1 and 4 during oxidation at pH 7. The replacement of the sixth ligand with methionine caused an increase in the reduction potential of the mutated heme of 222-244 mV. The midpoint potential of a triheme H52M cyt c 3 is higher than that of the wild type by approximately 50 mV, suggesting a contribution of the tetraheme architecture to the lowering of the reduction potentials. The hydrogen bonding of Thr24 with an axial ligand induces a decrease in reduction potential of approximately 50 mV. In conclusion, the bis-histidine coordination is strategically essential for the structure formation and the extremely low reduction potential of cyt c 3.

Ibuprofen Induces an Allosteric Conformational Transition in the Heme Complex of Human Serum Albumin with Significant Effects on Heme Ligation

Human serum albumin (HSA), the most prominent protein in blood plasma, is able to bind a wide range of endogenous and exogenous compounds. Among the endogenous ligands, HSA is a significant transporter of heme, the heme-HSA complex being present in blood plasma. Drug binding to heme-HSA affects allosterically the heme affinity for HSA and vice versa. Heme-HSA, heme, and their complexes with ibuprofen have been characterized by electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy. Comparison of the results for the heme and heme-HSA systems has provided insight into the structural consequences on the heme pocket of ibuprofen binding. The pentacoordinate tyrosine-bound heme coordination of heme-HSA, observed in the absence of ibuprofen, becomes hexacoordinate low spin upon ibuprofen binding, and heme dissociates at increasing drug levels. The electronic absorption spectrum and nu(Fe-CO)/nu(CO) vibrational frequencies of the CO-heme-HSA-ibuprofen complex, together with the observation of a Fe-His Raman mode at 218 cm(-1) upon photolysis of the CO complex and the low spin EPR g values indicate that a His residue is one of the low spin axial ligands, the sixth ligand probably being Tyr161. The only His residue in the vicinity of the heme Fe atom is His146, 9 A distant in the absence of the drug. This indicates that drug binding to heme-HSA results in a significant rearrangement of the heme pocket, implying that the conformational adaptability of HSA involves more than the immediate vicinity of the drug binding site. As a whole, the present spectroscopic investigation supports the notion that HSA could be considered as the prototype of monomeric allosteric proteins.

The Transcription Regulator RcoM-2 from Burkholderia xenovorans Is a Cysteine-Ligated Hemoprotein That Undergoes a Redox-Mediated Ligand Switch

Spectroscopic characterization of the newly discovered heme-PAS domain sensor protein BxRcoM-2 reveals that this protein undergoes redox-dependent ligand switching and CO- and NO-induced ligand displacement. The aerobic bacterium Burkholderia xenovorans expresses two homologous heme-containing proteins that promote CO-dependent transcription in vivo. These regulators of CO metabolism, BxRcoM-1 and BxRcoM-2, are gas-responsive heme-PAS domain proteins like mammalian neuronal PAS domain protein 2 (NPAS2) and the direct oxygen sensor from Escherichia coli ( EcDos). BxRcoM-2 was studied using electronic absorption, MCD, resonance Raman, and EPR spectroscopies. In the Fe(III) oxidation state, the heme is low-spin and six-coordinate with a cysteine(thiolate) as one of the two ligands. The sixth ligand is a histidine (His (74)), which is present in all states of the protein that were studied. Reduction to the Fe(II) oxidation state results in replacement of the cysteine(thiolate) with a neutral thioether ligand, Met (104). CO and NO bind to the Fe(II) BxRcoM-2 heme opposite the histidine ligand. Thus, BxRcoM-2 employs coordination state changes similar to those known for CO-sensing CooA, with redox-dependent loss of a cysteine(thiolate) ligand and displacement of a relatively weakly bound axial ligand by the effector gas molecule. Like EcDos, the weakly bound axial ligand that is displaced is methionine.

CYP56 (Dit2p) in Candida albicans : Characterization and Investigation of Its Role in Growth and Antifungal Drug Susceptibility

The complete DNA sequence of Candida albicans DIT2, encoding cytochrome P450 family 56 (CYP56), was obtained, and heterologous expression was achieved in Escherichia coli, where CYP56 was targeted to the membrane fraction. In reconstituted assays with the purified enzyme, CYP56 was shown to catalyze the conversion of N-formyl tyrosine into N,N'-bisformyl dityrosine, a reaction that was dependent on cytochrome P450 reductase, NADPH, and oxygen, yielding a turnover of 21.6 min(-1) and a k(s) of 26 microM. The Hill number was calculated as 1.6, indicating that two molecules of the substrate could bind to the protein. Azole antifungals could bind to the heme of CYP56 as a sixth ligand with high affinity. Both chromosomal alleles of CYP56 were disrupted using the SAT1 flipper technique, and CYP56 was found to be nonessential for cell viability under the culture conditions investigated. Susceptibility to azole drugs that bind to cytochromes P450 was tested, and the mutant showed unaltered susceptibility. However, the mutant showed increased susceptibility to the echinocandin drug caspofungin, suggesting an alteration in 1,3-glucan synthase and/or cell wall structure mediated by the presence of dityrosine. Phenotypically, the wild-type and mutant strains were morphologically similar when cultured in rich yeast extract-peptone-dextrose medium. However in minimal medium, the cyp56Delta mutant strain exhibited hyphal growth, in contrast to the wild-type strain, which grew solely in the yeast form. Furthermore, CYP56 was essential for chlamydospore formation.

Studies of the Carbon Dioxide and Epoxide Coupling Reaction in the Presence of Fluorinated Manganese(III) Acacen Complexes: Kinetics of Epoxide Ring-Opening

Five-coordinate manganese(III) complexes of N, N'-bis(trifluoroacetylacetone)-1,2-ethylenediimine (tfacacen) have been synthesized and structurally characterized by X-ray crystallography. The presence of the electron-withdrawing -CF3 substituents enhances the electrophilicity of the metal center in these (tfacacen)MnX (X=Cl, N3, NCO, NCS) derivatives when compared with their (acacen)MnX (acacen=N, N'-bis(acetylacetone)-1,2-ethylenediimine) analogs. This is demonstrated by the increased propensity of the Mn(III) center in the tfacacen complexes to bind a sixth ligand. Binding studies were performed utilizing the upsilonN3 stretching frequency in (tfacacen)MnN3, which is sensitive to the coordination of a ligand at the vacant axial site. Of importance, cyclohexene oxide was shown to readily bind to (tfacacen)MnN3, thereby providing an opportunity for directly monitoring the dependence of the epoxide ring-opening process on the metal complex concentration. In this instance, as has been amply demonstrated in the (salen)CrX case, the ring opening of cyclohexene oxide was found to be second-order in [(tfacacen)MnN3], with an activation energy of 71.0+/-6.0 kJ/mol. In the presence of strongly coordinating anions or amine bases, the rate of epoxide ring opening by (tfacacen)MnN3 was greatly retarded. The manganese cyanate and thiocyanate complexes were examined in an effort to develop other initiators for epoxide ring opening which provide readily accessible infrared spectroscopic probes. Indeed, the thiocyanate ligand was found to be well-suited for monitoring the epoxide ring-opening reaction by infrared spectroscopy.

Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum

Nitrate reductase from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harboring a bis(molybdopterin guanine dinucleotide) active site and a [4Fe-4S] cluster. Previous electron paramagnetic resonance (EPR) studies in both catalytic and inhibiting conditions showed that the molybdenum center has high coordination flexibility when reacted with reducing agents, substrates or inhibitors. As-prepared DdNapA samples, as well as those reacted with substrates and inhibitors, were crystallized and the corresponding structures were solved at resolutions ranging from 1.99 to 2.45 A. The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on that basis, the sixth molybdenum ligand, originally proposed to be an OH/OH(2) ligand, was assigned as a sulfur atom after refinement and analysis of the B factors of all the structures. This unexpected result was confirmed by a single-wavelength anomalous diffraction experiment below the iron edge (lambda = 1.77 A) of the as-purified enzyme. Furthermore, for six of the seven datasets, the S-S distance between the sulfur ligand and the Sgamma atom of the molybdenum ligand Cys(A140) was substantially shorter than the van der Waals contact distance and varies between 2.2 and 2.85 A, indicating a partial disulfide bond. Preliminary EPR studies under catalytic conditions showed an EPR signal designated as a turnover signal (g values 1.999, 1.990, 1.982) showing hyperfine structure originating from a nucleus of unknown nature. Spectropotentiometric studies show that reduced methyl viologen, the electron donor used in the catalytic reaction, does not interact directly with the redox cofactors. The turnover signal can be obtained only in the presence of the reaction substrates. With use of the optimized conditions determined by spectropotentiometric titration, the turnover signal was developed with (15)N-labeled nitrate and in D(2)O-exchanged DdNapA samples. These studies indicate that this signal is not associated with a Mo(V)-nitrate adduct and that the hyperfine structure originates from two equivalent solvent-exchangeable protons. The new coordination sphere of molybdenum proposed on the basis of our studies led us to revise the currently accepted reaction mechanism for periplasmic nitrate reductases. Proposals for a new mechanism are discussed taking into account a molybdenum and ligand-based redox chemistry, rather than the currently accepted redox chemistry based solely on the molybdenum atom.

Substrate Binding Triggers a Switch in the Iron Coordination in Dehaloperoxidase from Amphitrite ornata: HYSCORE Experiments

Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is the first known hemoglobin to exhibit efficient peroxidase activity in the oxidation of phenolic substrates. Hyperfine sublevel correlation spectroscopic (HYSCORE) analysis of the ferric form of DHP was carried out to characterize effects of the substrate 2,4,6-trifluorophenol (TFP) binding on the iron coordination in order to elucidate the molecular mechanism of the change in protein function from a globin to a peroxidase. Continuous wave EPR spectra show that heme iron of DHP at pH 6.0 exists in the high spin state. HYSCORE spectra recorded at magnetic field corresponding to g = 2 revealed the presence of exchangeable protons with hyperfine coupling of ca. 6 MHz, consistent with a water molecule being the sixth ligand in the iron coordination. These protons' spectral features disappeared upon substrate binding. This observation highlights the proposed role of the substrate as a trigger for the switch from hemoglobin to peroxidase function.

Electron Transfer and Electrocatalytic Properties of the Immobilized Methionine80Alanine Cytochrome c Variant

The M80A variant of yeast iso-1-cytochrome c (cytc), which features a noncoordinating Ala residue in place of the axial heme iron Met ligand, was chemisorbed on a gold electrode coated with 4-mercaptopyridine or carboxyalkanethiol self-assembled monolayers (SAM) and investigated by cyclic voltammetry at varying conditions of temperature, pH, and O2 concentration. The E degrees ' value (standard reduction potential for the heme Fe(III)/Fe(II) couple) of M80A cytc on both SAMs is of approximately -200 mV (vs the standard hydrogen electrode, SHE) at pH 7, which is more than 400 mV lower than that of native cytochrome c in the same conditions. The thermodynamics of Fe(III) to Fe(II) reduction and the kinetics of heterogeneous electron transfer (ET) are dominated by the presence of a hydroxide ion as the sixth axial heme iron ligand above pH 6. On both SAMs, protonation of the bound hydroxide ion is mainly responsible for the changes in these parameters at low pH, since the distances of ET between the heme and the electrode are found to be independent of pH in the range of 5-11. The invariance of the electrochemical features up to pH 11 indicates that no changes in heme iron coordination occur at high pH, at variance with native cytc. Most notably, immobilized M80A cytc is found to act as an efficient biocatalyst for O2 reduction from pH 5 to 11.0. This finding makes M80A cytc a suitable candidate as a constituent of a biocatalytic interface for O2 biosensing and opens the way for the exploitation of engineered cytochrome c in the bio-based detection of chemicals of environmental and clinical interest.