Conversion of a heme-based oxygen sensor to a heme oxygenase by hydrogen sulfide: effects of mutations in the heme distal side of a heme-based oxygen sensor phosphodiesterase (Ec DOS)

The heme-based oxygen-sensor phosphodiesterase from Escherichia coli (Ec DOS), is composed of an N-terminal heme-bound oxygen sensing domain and a C-terminal catalytic domain. Oxygen (O₂) binding to the heme Fe(II) complex in Ec DOS substantially enhances catalysis. Addition of hydrogen sulfide (H₂S) to the heme Fe(III) complex in Ec DOS also remarkably stimulates catalysis in part due to the heme Fe(III)–SH and heme Fe(II)–O₂ complexes formed by H₂S. In this study, we examined the roles of the heme distal amino acids, M95 (the axial ligand of the heme Fe(II) complex) and R97 (the O₂ binding site in the heme Fe(II)–O₂ complex) of the isolated heme-binding domain of Ec DOS (Ec DOS-PAS) in the binding of H₂S under aerobic conditions. Interestingly, R97A and R97I mutant proteins formed an oxygen-incorporated modified heme, verdoheme, following addition of H₂S combined with H₂O₂ generated by the reactions. Time-dependent mass spectroscopic data corroborated the findings. In contrast, H₂S did not interact with the heme Fe(III) complex of M95H and R97E mutants. Thus, M95 and/or R97 on the heme distal side in Ec DOS-PAS significantly contribute to the interaction of H₂S with the Fe(III) heme complex and also to the modification of the heme Fe(III) complex with reactive oxygen species. Importantly, mutations of the O₂ binding site of the heme protein converted its function from oxygen sensor to that of a heme oxygenase. This study establishes the novel role of H₂S in modifying the heme iron complex to form verdoheme with the aid of reactive oxygen species.     

Arg97 at the heme-distal side of the isolated heme-bound PAS domain of a heme-based oxygen sensor from Escherichia coli (Ec DOS) plays critical roles in autoxidation and binding to gases, particularly O2

The catalytic activity of heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) on cyclic di-GMP is markedly enhanced upon binding of gas molecules, such as O2 and CO, to the heme iron complex in the sensor domain. Arg97 interacts directly with O2 bound to Fe(II) heme in the crystal structure of the isolated heme-bound sensor domain with the PAS structure (Ec DOS-PAS) and may thus be critical in ligand recognition. To establish the specific role of Arg97, we generated Arg97Ala, Arg97Glu, and Arg97Ile mutant Ec DOS-PAS proteins and examined binding to O2, CO, and cyanide, as well as redox potentials. The autoxidation rates of the Arg97Ala and Arg97Glu mutant proteins were up to 2000-fold higher, while the O2 dissociation rate constant for dissociation from the Fe(II)-O2 heme complex of the Arg97Ile mutant was 100-fold higher than that of the wild-type protein. In contrast, the redox potential values of the mutant proteins were only slightly different from that of the wild type (within 10 mV). Accordingly, we propose that Arg97 plays critical roles in recognition of the O2 molecule and redox switching by stabilizing the Fe(II)-O2 complex, thereby anchoring O2 to the heme iron and lowering the autoxidation rate to prevent formation of Fe(III) hemin species not regulated by gas molecules. Arg97 mutations significantly influenced interactions with the internal ligand Met95, during CO binding to the Fe(II) complex. Moreover, the binding behavior of cyanide to the Fe(III) complexes of the Arg mutant proteins was similar to that of O2, which is evident from the Kd values, suggestive of electrostatic interactions between cyanide and Arg97.     

The roles of Tyr(CD1) and Trp(G8) in Mycobacterium tuberculosis truncated hemoglobin O in ligand binding and on the heme distal site architecture

The crystal structure of the cyano-met form of Mt-trHbO revealed two unusual distal residues Y(CD1) and W(G8) forming a hydrogen-bond network with the heme-bound ligand [Milani, M., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 5766-5771]. W(G8) is an invariant residue in group II and group III trHbs and has no counterpart in other globins. A previous study reported that changing Y(CD1) for a Phe causes a significant increase in the O2 combination rate, but almost no change in the O2 dissociation rate [Ouellet, H., et al. (2003) Biochemistry 42, 5764-5774]. Here we investigated the role of the W(G8) in ligand binding by using resonance Raman spectroscopy, stopped-flow spectrophotometry, and X-ray crystallography. For this purpose, W(G8) was changed, by site-directed mutagenesis, to a Phe in both the wild-type protein and the mutant Y(CD1)F to create the single mutant W(G8)F and the double mutant Y(CD1)F/W(G8)F, respectively. Resonance Raman results suggest that W(G8) interacts with the heme-bound O2 and CO, as evidenced by the increase of the Fe-O2 stretching mode from 559 to 564 cm-1 and by the lower frequency of the Fe-CO stretching modes (514 and 497 cm-1) compared to that of the wild-type protein. Mutation of W(G8) to Phe indicates that this residue controls ligand binding, as evidenced by a dramatic increase of the combination rates of both O2 and CO. Also, the rate of O2 dissociation showed a 90-1000-fold increase in the W(G8)F and Y(CD1)F/W(G8)F mutants, that is in sharp contrast with the values obtained for the other distal mutants Y(B10)F and Y(CD1)F [Ouellet, H., et al. (2003) Biochemistry 42, 5764-5774]. Taken together, these data indicate a pivotal role for the W(G8) residue in O2 binding and stabilization.     

Role of Phe113 at the distal side of the heme domain of an oxygen-sensor (Ec DOS) in the characterization of the heme environment

The heme-based oxygen-sensor enzyme from Escherichia coli (Ec DOS) is a heme-regulated phosphodiesterase with activity on cyclic-di-GMP and is composed of an N-terminal heme-bound sensor domain with the PAS structure and a C-terminal functional domain. The activity of Ec DOS is substantially enhanced by the binding of O₂ to the Fe(II)-protoporphyrin IX complex [Fe(II) complex] in the sensor domain. The binding of O₂ to the Fe(II) complex changes the structure of the sensor domain, and this altered structure becomes a signal that is transduced to the functional domain to trigger catalysis. The first step in intra-molecular signal transduction is the binding of O₂ to the Fe(II) complex, and detailed elucidation of this molecular mechanism is thus worthy of exploration. The X-ray crystal structure reveals that Phe113 is located close to the O₂ molecule bound to the Fe(II) complex in the sensor domain. Here, we found that the O₂ association rate constants (>200 × 10⁻³ μM⁻¹ s⁻¹: F113L; 26 × 10⁻³ μM⁻¹ s⁻¹: F113Y) of the Fe(II) complexes of Phe113 mutants were markedly different from that (51 × 10⁻³ μM⁻¹ s⁻¹) of the wild-type enzyme, and auto-oxidation rates (0.00068 min⁻¹: F113L; 0.039 min⁻¹: F113Y) of the Phe113 mutants also differed greatly from that (0.0062 min⁻¹) of the wild-type enzyme. We thus suggest that Phe113, residing near the O₂ molecule, has a critical role in optimizing the Fe(II)-O₂ complex for effective regulation of catalysis by the oxygen-sensor enzyme. Interactions of CO and cyanide anion with the mutant proteins were also studied.     

Unique heme environment at the putative distal region of hydrogen peroxide-dependent fatty acid alpha-hydroxylase from Sphingomonas paucimobilis (peroxygenase P450(SPalpha)

Fatty acid alpha-hydroxylase from Sphingomonas paucimobilis is a hydrogen peroxide-dependent cytochrome P450 (P450) enzyme (P450(SPalpha)). In this study, heme-ligand exchange reactions of P450(SPalpha) were investigated using the optical spectroscopic method and compared with those of various P450s. Alkylamines (C >/= 5) induced changes in the spectrum of ferric P450(SPalpha) to one typical of a nitrogenous ligand-bound low-spin form of ferric P450, although their affinities were lower than those for other P450s, and a substrate, laurate, did not interfere with the binding in contrast with in the cases of other P450s. Other compounds having a nitrogen donor atom to the heme iron of P450, including pyridine or 1-methylimidazole, induced no change in the spectrum of P450(SPalpha) in either the ferric or ferrous state. Practically no spectral change was observed on the addition of alkyl isocyanides to ferric P450s. On the other hand, cyanide induced a change in the spectrum of ferric P450(SPalpha) to one characteristic of cyanide-bound form of ferric P450. The affinity of cyanide increased when the substrate was added, in contrast with in the cases of other P450s. Ferrous P450(SPalpha) combined with CO and alkyl isocyanides, and the affinity for CO was of the same order of magnitude as in the cases of other P450s. These findings suggest a unique heme environment of P450(SPalpha), in which most compounds usually acting as external ligands of ferric P450s are prevented from gaining access to the heme iron of P450(SPalpha). The unique properties of the hydroxylase reaction catalyzed by P450(SPalpha), where an oxygen atom of hydrogen peroxide but not of molecular oxygen is utilized and incorporated into a fatty acid at its alpha position, is possibly related with such a specific heme environment of this P450. A possible mechanism for the peroxygenase reaction of P450(SPalpha) is proposed.     

Relative stability of the S2 isomers of the oxygen evolving complex of photosystem II

Photosynthesis Research - The oxidation of water to O2 is catalyzed by the Oxygen Evolving Complex (OEC), a Mn4CaO5 complex in Photosystem II (PSII). The OEC is sequentially oxidized from state S0...     

Critical roles of Leu99 and Leu115 at the heme distal side in auto-oxidation and the redox potential of a heme-regulated phosphodiesterase from Escherichia coli

The heme-regulated phosphodiesterase from Escherichia coli (Ec DOS), which is a heme redox-dependent enzyme, is active with a ferrous heme but inactive with a ferric heme. Global structural changes including axial ligand switching and a change in the rigidity of the FG loop accompanying the heme redox change may be related to the dependence of Ec DOS activity on the redox state. Axial ligands such as CO, NO, and O2 act as inhibitors of Ec DOS because they interact with the ferrous heme complex. The X-ray crystal structure of the isolated heme-bound domain (Ec DosH) shows that Leu99, Phe113 and Leu115 indirectly and directly form a hydrophobic triad on the heme plane and that they should be located at or near the ligand access channel of the heme iron. We generated L99T, L99F, L115T, and L115F mutants of Ec DosH and examined their physicochemical characteristics, including auto-oxidation rates, O2 and CO binding kinetics, and redox potentials. The Fe(III) complex of the L115F mutant was unstable and had a Soret absorption spectrum located 5 nm lower than those of the wild-type and other mutants. Auto-oxidation rates of the mutants (0.049-0.33 min(-1)) were much higher than that of the wild-type (0.0063 min(-1)). Furthermore, the redox potentials of the former three mutants (23.1-34.6 mV versus SHE) were also significantly lower than that of the wild-type (63.9 mV versus SHE). Interaction between O2 and the L99F mutant was different from that in the wild-type, whereas CO binding rates of the mutants were similar to those of the wild-type. Thus, it appears that Leu99 and Leu115 are critical for determining the characteristics of heme iron. Finally, we discuss the roles of these amino-acid residues in the heme electronic states.     

Generation of reactive oxygen species by photolysis of the ruthenium(II) complex Ru(NH3)5(pyrazine)2+ in oxygenated solution.

Metal-to-ligand charge transfer photolysis of the ruthenium(II) pyrazine complex Ru(NH3)5pz2+ (I) in pH 7.4 oxygenated phosphate buffer solution generates the Ru(III) analog Ru(NH3)5pz3+ plus the reactive oxygen species singlet oxygen and superoxide. Based on the very short MLCT lifetime (re-measured as approximately 250 ps in D2O) of I* and the quantum yield for singlet oxygen formation (0.01 for aerated D2O) the rate constant for oxygen quenching of I* was calculated to be approximately (3+/-1)x10(10) M-1 s-1.     

Crystal Structure of the 2-Oxoglutarate- and Fe(II)-Dependent Lysyl Hydroxylase JMJD6

Lysyl and prolyl hydroxylations are well-known post-translational modifications to animal and plant proteins with extracellular roles. More recent work has indicated that the hydroxylation of intracellular animal proteins may be common. JMJD6 catalyses the iron- and 2-oxoglutarate-dependent hydroxylation of lysyl residues in arginine-serine-rich domains of RNA-splicing-related proteins. We report crystallographic studies on the catalytic domain of JMJD6 in complex with Ni(II) substituting for Fe(II). Together with mutational studies, the structural data suggest how JMJD6 binds its lysyl residues such that it can catalyse C-5 hydroxylation rather than N^?-demethylation, as for analogous enzymes.     

Strand opening by the UvrA(2)B complex allows dynamic recognition of DNA damage.

Repair proteins alter the local DNA structure during nucleotide excision repair (NER). However, the precise role of DNA melting remains unknown. A series of DNA substrates containing a unique site-specific BPDE-guanine adduct in a region of non-complementary bases were examined for incision by the Escherichia coli UvrBC endonuclease in the presence or absence of UvrA. UvrBC formed a pre-incision intermediate with a DNA substrate containing a 6-base bubble structure with 2 unpaired bases 5' and 3 unpaired bases 3' to the adduct. Formation of this bubble served as a dynamic recognition step in damage processing. UvrB or UvrBC may form one of three stable repair intermediates with DNA substrates, depending upon the state of the DNA surrounding the modified base. The dual incisions were strongly determined by the distance between the adduct and the double-stranded-single-stranded DNA junction of the bubble, and required homologous double-stranded DNA at both incision sites. Remarkably, in the absence of UvrA, UvrBC nuclease can make both 3' and 5' incisions on substrates with bubbles of 3-6 nucleotides, and an uncoupled 5' incision on bubbles of >/=>/=10 nucleotides. These data support the hypothesis that the E.coli and human NER systems recognize and process DNA damage in a highly conserved manner.     

Importance of the G27-A43 mismatch at the anticodon stem of Escherichia coli tRNA(Thr2)

The tRNA(Thr2) isoacceptor of E. coli has a G-A mismatch at positions 27-43. When the anticodon of this tRNA was converted to an amber anticodon (CUA), this tRNA showed suppressor activity in E. coli. Moreover, introduction of the base pair (G-C or U-A) at positions 27-43 of this suppressor tRNA reduced its suppressor activity. These results indicate that the G27-A43 mismatch is necessary for full function of tRNA(Thr2).     

Sequence-specific deoxyribonucleic acid (DNA) recognition by steroidogenic factor 1: a helix at the carboxy terminus of the DNA binding domain is necessary for complex stability

Steroidogenic factor 1 (SF1) is a member of the NR5A subfamily of nuclear hormone receptors and is considered a master regulator of reproduction because it regulates a number of genes encoding reproductive hormones and enzymes involved in steroid hormone biosynthesis. Like other NR5A members, SF1 harbors a highly conserved approximately 30-residue segment called the FTZ-F1 box C-terminal to the core DNA binding domain (DBD) common to all nuclear receptors and binds to 9-bp DNA sequences as a monomer. Here we describe the solution structure of the SF1 DBD in complex with an atypical sequence in the proximal promoter region of the inhibin-alpha gene that encodes a subunit of a reproductive hormone. SF1 forms a specific complex with the DNA through a bipartite motif binding to the major and minor grooves through the core DBD and the N-terminal segment of the FTZ-F1 box, respectively, in a manner previously described for two other monomeric receptors, nerve growth factor-induced-B and estrogen-related receptor 2. However, unlike these receptors, SF1 harbors a helix in the C-terminal segment of the FTZ-F1 box that interacts with both the core DBD and DNA and serves as an important determinant of stability of the complex. We propose that the FTZ-F1 helix along with the core DBD serves as a platform for interactions with coactivators and other DNA-bound factors in the vicinity.     

Regulation of Photosystem II core protein phosphorylation at the substrate level: Light induces exposure of the CP43 chlorophyll a protein complex to thylakoid protein kinase(s)

Light induces conformational changes in the CP43 chl-a-protein antenna complex in isolated PS II core-complexes exposing phosphorylation site(s) to PS II core-associated protein kinase(s), to added solubilized thylakoid protein kinase(s), as well as to tryptic cleavage. The substrate-activation effect is demonstrated by exposure of the PS II cores to light during the kinase assay as well as by preillumination of the PS II cores in which the endogenous kinase(s) has been inactivated by treatment with N-ethylmaleimid. In the latter case, phosphorylation was performed in darkness following addition of the solubilized protein kinase(s). The solubilized protein kinase(s) does not require light activation. The apparent molecular masses of the main protein kinase(s) associated with the PS II cores (about 31–35 kDa and 45 kDa) differ from that of the major protein kinase present in solubilized preparations obtained from spinach thylakoids (64 kDa). The light-induced exposure of CP43 increases with the light intensity in the range of 20–100 μmol photons m⁻² s⁻¹ as demonstrated by preillumination of N-ethylmaleimid treated cores followed by addition of the solubilized protein kinase(s) and performing the phosphorylation assay in darkness. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ligand dynamics controlled reverse spin cross over in bis pyrazolyl pyridine based Fe(II) complex cation with metallodithiolato anions with an example of a ferromagnetic 2:1 cocrystal of mixed Ni(III)/Ni(II) oxidation states

We report here the crystal and molecular structures of three compounds [FeL₂] [Ni(mnt)₂] (1), [FeL₂]₂ [Ni(mnt)₂]₃·2H₂O (2) and [FeL₂] [Cu(mnt)₂]·2CH₃CN (3) where L = 2,6-bis(3,5-dimethylpyrazol-1-ylmethyl)pyridine and mnt = maleonitriledithiolate, and their detailed spectroscopic and magnetic properties using variable temperature Mössbauer, EPR, susceptibility studies, along with room temperature electron spectroscopy for chemical analysis (ESCA) studies. The observed temperature dependant high spin/low spin (HS/LS) ratios of [FeL₂]²⁺ cations in these lattices, exhibiting ‘reverse spin cross-over’ measured unequivocally by Mössbauer, have been interpreted as resulting from differing amount of ‘void space’ in the lattice, a measure of the ease of lattice dynamics originating from ligand L. Differential scanning calorimetric data points this HS/LS transition to order-disorder type of second order phase transitions. While trying to test this lattice dynamics controlled property of [FeL₂]²⁺ cations an unusual behavior of cocrystallization of two planar complex anions of the same type in two different oxidation states, viz. [Ni(mnt)₂]²⁻ and [Ni(mnt)₂]⁻, was observed in [FeL₂]₂ [Ni(mnt₂)]₃, supported by crystallography, ESCA chemical shifts of Ni 2p_3/2 and EPR. The susceptibility data in combination with ESCA chemical shifts of S 2p_3/2 and Ni 2p_3/2 on all the compounds reveal the importance of charge transfer between the two counter ions.     

Copper(II) complexes of a nonsteroidal anti-inflammatory drug niflumic acid. Synthesis, crystal structure of tetrakis-mu-(2-[3-(trifluoromethyl) phenyl]aminonicotinato)bis(dimethylsulfoxide)-dicopper(II) complex at 190 K. Anti-inflammatory properties.

The synthesis and characterization of three complexes with a potent nonsteroidal anti-inflammatory drug niflumic acid {2-[3-(trifluoromethyl)phenyl]aminonicotinic acid} with formula [Cu(niflumato)2L] (L = H2O, DMSO = dimethylsulfoxide, DMF = N,N-dimethylformamide) were investigated. The crystal and molecular structure of the {Cu(niflumato)2(DMSO)}2 was reported. Crystallographic data are as follows: monoclinic system, space group P2(1)/n, Z = 2, a = 11.1318(8), b = 17.513(2), c = 15.336(1) A, beta = 103.316(8) degrees, V = 2909.4(4) A3. The structure was refined to R = 0.030 and wR = 0.037 for 3702 reflections with I > sigma (I). It consists of centrosymmetric binuclear units with the Cu-Cui (symmetry code i: 1-x, -y, 1-z) distance between two centrosymmetrically related ions of 2.6272(5) A. Each Cu(II) ion in [Cu2(DMSO)2(mu-niflumato)4] is coordinated to an apical dimethylsulfoxide O atom on the one hand and to the equatorial carbonyl and carboxylic O atoms of two crystallographically independent niflumate moieties and their centrosymmetric counterparts on the other hand. In spite of the low-temperature (190 K) crystal measurements, one L-CF3 grouping exhibits some disorder. The biological activities of these complexes were compared to that of niflumic acid. Niflumic acid and its various copper complexes significantly inhibited polymorphonuclear leukocyte (PMNL) oxidative metabolism, as assessed by chemiluminescence and O2- generation measurement. This effect was dose-dependent. All copper complexes exerted a similar inhibiting effect which was always significantly higher than that exerted by the parent drug.     

Electronic absorption spectroscopy and time-dependent density functional theory calculations on the nickel(II) complex of 1,4-bis(pyrrol-2-ylmethyleneamino)butane

The electronic structure of the 1,4-bis(pyrrol-2-ylmethyleneamino)butane nickel(II) complex has been studied using electronic absorption spectroscopy and density functional theory (DFT) calculations. The DFT optimised structure is in excellent agreement with the X-ray crystal structure of the complex and time-dependent DFT calculations have been used to probe the nature of the transitions observed in the electronic absorption spectrum.     

AP2 clathrin adaptor complex, but not AP1, controls the access of the major histocompatibility complex (MHC) class II to endosomes

Newly synthesized MHC II alpha- and beta-chains associated with the invariant chain chaperone (Ii) enter the endocytic pathway for Ii degradation and loading with peptides before transport to the cell surface. It is unclear how alphabetaIi complexes are sorted from the Golgi apparatus and directed to endosomes. However, indirect evidence tends to support direct transport involving the AP1 clathrin adaptor complex. Surprisingly, we show here that knocking down the production of AP1 by RNA interference did not affect the trafficking of alphabetaIi complexes. In contrast, AP2 depletion led to a large increase in surface levels of alphabetaIi complexes, inhibited their rapid internalization, and strongly delayed the appearance of mature MHC II in intracellular compartments. Thus, in the cell systems studied here, rapid internalization of alphabetaIi complexes via an AP2-dependent pathway represents a key step for MHC II delivery to endosomes and lysosomes.     

Effects of NO2-modification of Tyr83 on the reactivity of spinach plastocyanin with inorganic redox partners [Fe(CN)6]3-/4- and [Co(phen)3]3+/2+

Plastocyanin (PCu) from spinach leaves has been singly NO2-modified, purified by FPLC, and the position of modification at Tyr83 confirmed by trypsin digestion and amino-acid sequencing. Electron-transfer reactions of native and NO2-modified PCu with the inorganic redox partners [Fe(CN)6]3- and [Co(phen)3]3+, as oxidants for PCu(I), and [Fe(CN)6]4- and [Co(phen)3]2+ as reductants for PCu(II), have been studied as a function of pH. The acid dissociation constant for the phenolic group on NO2-Tyr83 PCu is 8.78 (average) for reduced, and 8.10 for oxidised protein, as compared to values greater than 10 for native protein. At I = 0.10 M (NaCl) NO2-modification brings about a 20 mV increase in reduction potential at pH less than 7 and deprotonation of the phenolic group a 20-25 mV decrease, both transmitted to and effective at the active site. Deprotonation brings about a 48% increase in rate for [Fe(CN)6]3- and a 47% decrease for [Fe(CN)6]4- in accordance with these changes. In the case of [Co(phen)3]3+, which reacts substantially at the remote site in the vicinity of Tyr83, the influence of deprotonation on the active site is supplemented by the negative charge of the phenolate, and a total increase of 131% is observed. These results can be understood on the basis of the electron-transfer theory, and add support to the belief that electron transfer kinetics of negatively and positively charged reactants are dominated by different sites on PCu for electron transfer, namely adjacent (close to His87) and remote (close to Tyr83), respectively.