Appending a tris-imidazole ligand with a Tyr 244 mimic on the distal face of bromoacetamidoporphyrin

[structure: see text] Bromoacetamidoporphyrin is a convenient synthon for the attachment of distal superstructures at room temperature in good yields. New models are presented that contain a tris-imidazole distal ligand set bound to the porphyrin in either a binary or trinary fashion. More importantly, one distal imidazole is cross-linked to a phenol mimicking Tyr(244), making this model the closest structural analogue yet reported of the metal free cytochrome c oxidase (CcO) active site.     

Insights into the mechanism of proton transport in cytochrome c oxidase

Cytochrome c oxidase (CcO), known as complex IV of the electron transport chain, plays several important roles in aerobic cellular respiration. Electrons transferred from cytochrome c to CcO's catalytic site reduce molecular oxygen and produce a water molecule. These electron transfers also drive active proton pumping from the matrix (N-side) to intermembrane region (P-side) in mitochondria; the resultant proton gradient activates ATP synthase to produce ATP from ADP. Although the existence of the coupling between the electron transfer and the proton transport (PT) is established experimentally, its mechanism is not yet fully understood at the molecular level. In this work, it is shown why the reduction of heme a is essential for proton pumping. This is demonstrated via novel reactive molecular dynamics (MD) simulations that can describe the Grotthuss shuttling associated with the PT as well as the dynamic delocalization of the excess proton electronic charge defect. Moreover, the "valve" role of the Glu242 residue (bovine CcO notation) and the gate role of d-propionate of heme a(3) (PRDa3) in the explicit PT are explicitly demonstrated for the first time. These results provide conclusive evidence for the CcO proton transporting mechanism inferred from experiments, while deepening the molecular level understanding of the CcO proton switch.     

NO and O2 reactivities of synthetic functional models of nitric oxide reductase and cytochrome c oxidase

Though nitric oxide reductase (NOR) and cytochrome c oxidase (CcO) have similar active sites, they exhibit quite different functions. While NOR reduces NO to N(2)O, CcO reduces O(2) to H(2)O. Further, CcO is reversibly inhibited by NO, the substrate for NOR, and NOR is reversibly inhibited by O(2), which is the substrate for CcO. Over the past decade several structural and functional models of these enzymes have been reported. The mimics have been used to understand the reaction mechanism of these enzymes and develop structure function correlations for these active sites. This article summarizes these recent developments with particular stress on the reactivities of functional mimics of CcO with NO and functional mimics of NOR with O(2).     

Acidity of a Cu-bound histidine in the binuclear center of cytochrome C oxidase

Cytochrome c oxidase (CcO) is a crucial enzyme in the respiratory chain. Its function is to couple the reduction of molecular oxygen, which takes place in the Fea3-CuB binuclear center, to proton translocation across the mitochondrial membrane. Although several high-resolution structures of the enzyme are known, the molecular basis of proton pumping activation and its mechanism remain to be elucidated. We examine a recently proposed scheme (J. Am. Chem. Soc. 2004, 126, 1858; FEBS Lett. 2004, 566, 126) that involves the deprotonation of the CuB-bound imidazole ring of a histidine (H291 in mammalian CcO) as a key element in the proton pumping mechanism. The central feature of that proposed mechanism is that the pKa values of the imidazole vary significantly depending on the redox state of the metals in the binuclear center. We use density functional theory in combination with continuum electrostatics to calculate the pKa values, successively in bulk water and within the protein, of the Cu-bound imidazole in various Cu- and Cu-Fe complexes. From pKas in bulk water, we derived a value of -266.34 kcal.mol(-1) for the proton solvation free energy (Delta). This estimate is in close agreement with the experimental value of -264.61 kcal.mol(-1) (J. Am. Chem. Soc. 2001, 123, 7314), which reinforces the conclusion that Delta is more negative than previous values used for pKa calculations. Our approach, on the basis of the study of increasingly more detailed models of the CcO binuclear center at different stages of the catalysis, allows us to examine successively the effect of each of the two metals' redox states and of solvation on the acidity of imidazole, whose pKa is approximately 14 in bulk water. This analysis leads to the following conclusions: first, the effect of Cu ligation on the imidazole acidity is negligible regardless of the redox state of the metal. Second, results obtained for Cu-Fe complexes in bulk water indicate that Cu-bound imidazole pKa values lie within the range of 14.8-16.6 throughout binuclear redox states corresponding to the catalytic cycle, demonstrating that the effect of the Fe oxidation states is also negligible. Finally, the low-dielectric CcO proteic environment shifts the acid-base equilibrium toward a neutral imidazole, further increasing the corresponding pKa values. These results are inconsistent with the proposed role of the Cu-bound histidine as a key element in the pumping mechanism. Limitations of continuum solvation models in pKa calculations are discussed.     

Luminescent ruthenium(II)- and rhenium(I)-diimine wires bind nitric oxide synthase

Ru(II)- and Re(I)-diimine wires bind to the oxygenase domain of inducible nitric oxide synthase (iNOSoxy). In the ruthenium wires, [Ru(L)2L']2+, L' is a perfluorinated biphenyl bridge connecting 4,4'-dimethylbipyridine to a bulky hydrophobic group (adamantane, 1), a heme ligand (imidazole, 2), or F (3). 2 binds in the active site of the murine iNOSoxy truncation mutants Delta65 and Delta114, as demonstrated by a shift in the heme Soret from 422 to 426 nm. 1 and 3 also bind Delta65 and Delta114, as evidenced by biphasic luminescence decay kinetics. However, the heme absorption spectrum is not altered in the presence of 1 or 3, and Ru-wire binding is not affected by the presence of tetrahydrobiopterin or arginine. These data suggest that 1 and 3 may instead bind to the distal side of the enzyme at the hydrophobic surface patch thought to interact with the NOS reductase module. Complexes with properties similar to those of the Ru-diimine wires may provide an effective means of NOS inhibition by preventing electron transfer from the reductase module to the oxygenase domain. Rhenium-diimine wires, [Re(CO)3L1L1']+, where L1 is 4,7-dimethylphenanthroline and L1' is a perfluorinated biphenyl bridge connecting a rhenium-ligated imidazole to a distal imidazole (F8bp-im) (4) or F (F9bp) (5), also form complexes with Delta114. Binding of 4 shifts the Delta114 heme Soret to 426 nm, demonstrating that the terminal imidazole ligates the heme iron. Steady-state luminescence measurements establish that the 4:Delta114 dissociation constant is 100 +/- 80 nM. Re-wire 5 binds Delta114 with a K(d) of 5 +/- 2 microM, causing partial displacement of water from the heme iron. Our finding that both 4 and 5 bind in the NOS active site suggests novel designs for NOS inhibitors. Importantly, we have demonstrated the power of time-resolved FET measurements in the characterization of small molecule:protein interactions that otherwise would be difficult to observe.     

Distal metal effects in cobalt porphyrins related to CcO

Cobalt(II) porphyrins were studied to determine the influence of distal site metalation and superstructure upon dioxygen reactivity in active site models of cytochrome c oxidase (CcO). Monometallic, Co(II)(P) complexes when ligated by an axial imidazole react with dioxygen to form reversible Co-superoxide adducts, which were characterized by EPR and resonance Raman (RR). Unexpectedly, certain Co porphyrins with Cu(I) metalated imidazole pickets do not form mu-peroxo Co(III)/Cu(II) products even though the calculated intermetallic distance suggests this is possible. Instead, cobalt-porphyrin-superoxide complexes are obtained with the distal copper remaining as Cu(I). Moreover, distal metals (Cu(I) or Zn(II)) greatly enhance the stability of the dioxygen adduct, such that Co superoxides of bimetallic complexes demonstrate minimal reversibility. The "trapping" of dioxygen by a second metal is attributed to structural and electrostatic changes within the distal pocket upon metalation. EPR evidence suggests that the terminal oxygen in these bimetallic Co-superoxide systems is H-bonded to the NH of an imidazole picket amide linker, which may contribute to enthalpic stabilization of the dioxygen adduct. Stabilization of the dioxygen adduct in these bimetallic systems suggests one possible role for the distal copper in the Fe/Cu bimetallic active site of terminal oxidases, which form a heme-superoxide/copper(I) adduct upon oxygenation.     

Photoreactions of cytochrome C oxidase

The photoreduction of oxidized bovine heart cytochrome c oxidase (CcO) by visible and UV radiation was investigated in the absence and presence of external reagents. In the former case, the quantum yields for direct photoreduction of heme A (heme a + heme a(3)) were 2.6 +/- 0.5 x 10(-3), 4 +/- 1 x 10(-4), and 4 +/- 2 x 10(-6) with pulsed laser irradiation at 266, 355 and 532 nm, respectively. Within experimental uncertainty, the quantum yields did not depend on pulse energy, implying that the mechanism is monophotonic. Irradiation with 355 nm light resulted in spectral changes similar to those produced independently by reduction with dithionite, whereby the low-spin heme a and Cu(A) are reduced first. Extended illumination at 355 and 532 nm yielded substantial amounts of reduced heme a(3). Heme decomposition was noted with 266 nm light. In the presence of formate and cyanide ions, which bind at the binuclear heme a(3)/copper center in CcO, irradiation at 355 nm caused selective reduction of only the low-spin heme a and Cu(A). The addition of ferrioxalate ion dramatically increased the efficiency of cytochrome c oxidase photoreduction. The quantum efficiency for heme A reduction was found to be near unity, significantly greater than for other known methods of photoreduction. The active reductant is most likely ferrous iron, and its reduction of the enzyme is thermodynamically driven by the reformation of ferrioxalate in the presence of excess oxalate ion. Other metalloenzymes with redox potentials similar to those of cytochrome c oxidase should be amenable to indirect photoreduction by this method.     

Direct determination of the complete set of iron normal modes in a porphyrin-imidazole model for carbonmonoxy-heme proteins: [Fe(TPP)(CO)(1-MeIm)

Detailed Fe vibrational spectra have been obtained for the heme model complex [Fe(TPP)(CO)(1-MeIm)] using a new, highly selective and quantitative technique, Nuclear Resonance Vibrational Spectroscopy (NRVS). This spectroscopy measures the complete vibrational density of states for iron atoms, from which normal modes can be calculated via refinement of the force constants. These data and mode assignments can reveal previously undetected vibrations and are useful for validating predictions based on optical spectroscopies and density functional theory, for example. Vibrational modes of the iron porphyrin-imidazole compound [Fe(TPP)(CO)(1-MeIm)] have been determined by refining normal mode calculations to NRVS data obtained at an X-ray synchrotron source. Iron dynamics of this compound, which serves as a useful model for the active site in the six-coordinate heme protein, carbonmonoxy-myoglobin, are discussed in relation to recently determined dynamics of a five-coordinate deoxy-myoglobin model, [Fe(TPP)(2-MeHIm)]. For the first time in a six-coordinate heme system, the iron-imidazole stretch mode has been observed, at 226 cm(-)(1). The heme in-plane modes with large contributions from the nu(42), nu(49), nu(50), and nu(53) modes of the core porphyrin are identified. In general, the iron modes can be attributed to coupling with the porphyrin core, the CO ligand, the imidazole ring, and/or the phenyl rings. Other significant findings are the observation that the porphyrin ring peripheral substituents are strongly coupled to the iron doming mode and that the Fe-C-O tilting and bending modes are related by a negative interaction force constant.     

Significant improvement of oxidase activity through the genetic incorporation of a redox-active unnatural amino acid

While nature employs various covalent and non-covalent strategies to modulate tyrosine (Y) redox potential and pK _a in order to optimize enzyme activities, such approaches have not been systematically applied for the design of functional metalloproteins. Through the genetic incorporation of 3-methoxytyrosine (OMeY) into myoglobin, we replicated important features of cytochrome c oxidase (CcO) in this small soluble protein, which exhibits selective O₂ reduction activity while generating a small amount of reactive oxygen species (ROS). These results demonstrate that the electron donating ability of a tyrosine residue in the active site is important for CcO function. Moreover, we elucidated the structural basis for the genetic incorporation of OMeY into proteins by solving the X-ray structure of OMeY specific aminoacyl-tRNA synthetase complexed with OMeY.     

Spectroscopic evidence for a heme-superoxide/Cu(I) intermediate in a functional model of cytochrome c oxidase

A superstructured tetraphenylporphyrin with a covalently attached proximal imidazole axial base and three distal imidazole pickets has been developed as a model for the active site of terminal oxidases such as cytochrome c oxidase. The oxygen adduct of the Fe-only heme (at low temperature) has a diamagnetic NMR and is EPR silent, which taken together with a resonance Raman oxygen isotope sensitive band (nuFe-O) at 575/554 cm-1 (16O2/18O2) indicates formation of a six-coordinate heme-superoxide complex. Unexpectedly, the Fe/Cu complex, where the copper is in a trisimidazole environment approximately 5 A above the heme plane, displays similar characteristics: a diamagnetic NMR, EPR silence, and nuFe-O at 570/544 cm-1. This indicates the dioxygen adduct of this Fe/Cu system is also a superoxide. This contrasts with previously characterized partially reduced dioxygen intermediates of binuclear heme/copper complexes that form Fe/Cu mu-peroxo complexes.     

Picosecond photoreduction of inducible nitric oxide synthase by rhenium(I)-diimine wires

In a continuing effort to unravel mechanistic questions associated with metalloenzymes, we are developing methods for rapid delivery of electrons to deeply buried active sites. Herein, we report picosecond reduction of the heme active site of inducible nitric oxide synthase bound to a series of rhenium-diimine electron-tunneling wires, [Re(CO)3LL']+, where L is 4,7-dimethylphenanthroline and L' is a perfluorinated biphenyl bridge connecting a rhenium-ligated imidazole or aminopropylimidazole to a distal imidazole (F8bp-im (1) and C3-F8bp-im (2)) or F (F9bp (3) and C3-F9bp (4)). All four wires bind tightly (Kd in the micromolar to nanomolar range) to the tetrahydrobiopterin-free oxidase domain of inducible nitric oxide synthase (iNOSoxy). The two fluorine-terminated wires displace water from the active site, and the two imidazole-terminated wires ligate the heme iron. Upon 355-nm excitation of iNOSoxy conjugates with 1 and 2, the active site Fe(III) is reduced to Fe(II) within 300 ps, almost 10 orders of magnitude faster than the naturally occurring reduction.     

Probing heme coordination states of inducible nitric oxide synthase with a ReI(imidazole-alkyl-nitroarginine) sensitizer-wire

Mammalian inducible nitric oxide synthase (iNOS) catalyzes the production of l-citrulline and nitric oxide (NO) from L-arginine and O2. The Soret peak in the spectrum of the iNOS heme domain (iNOSoxy) shifts from 423 to 390 nm upon addition of a sensitizer-wire, [ReI-imidazole-(CH2)8-nitroarginine]+, or [ReC8argNO2]+, owing to partial displacement of the water ligand in the active site. From analysis of competitive binding experiments with imidazole, the dissociation constant (Kd) for [ReC8argNO2]+-iNOSoxy was determined to be 3.0+/-0.1 microM, confirming that the sensitizer-wire binds with higher affinity than both L-arginine (Kd=22+/-5 microM) and imidazole (Kd=14+/-3 microM). Laser excitation (355 nm) of [ReC8argNO2]+-iNOSoxy triggers electron transfer to the active site of the enzyme, producing a ferroheme in less than approximately 1 micros.     

Quantum chemical calculations of the NHA bound nitric oxide synthase active site: O2 binding and implications for the catalytic mechanism

Density functional theory has been employed to model the binding of the intermediate substrate NHA, by nitric oxide synthases. In particular, the orientation and interactions of possibly catalytically important substrate hydrogens, with and without molecular oxygen bound to the active site heme group, are considered. Without O(2), three possible conformers have been found, with the energetically most favored structure being that in which both protons of the -NHOH moiety of NHA are directed toward the heme group. With oxygen bound, four different structures were found. The energetically lowest structure is again found to have both hydrogens of the -NHOH group pointing toward the heme group, thus forming hydrogen bonds between -NH- and the terminal oxygen, and between -OH and the inner oxygen of the heme-O(2) group. In addition, unprotonated structures of the substrate bound to the active site are considered and the proton affinity calculated.     

DFT/electrostatic calculations of pK(a) values in cytochrome c oxidase

Using classical electrostatic calculations, earlier we examined the dependence of the protonation state of bovine cytochrome c oxidase (CcO) on its redox state. Based on these calculations, we have proposed a model of CcO proton pumping that involves His291, one of the Cu(B) histidine ligands, which was found to respond to redox changes of the enzyme Fe(a)(3)-Cu(B) catalytic center. In this work, we employ combined density functional and continuum electrostatic calculations to evaluate the pK(a)() values of His291 and Glu242, two key residues of the model. The pK(a) values are calculated for different redox states of the enzyme, and the influence of different factors on the pK(a)'s is analyzed in detail. The calculated pK(a)() values of Glu242 are between 9.4 and 12.0, depending on the redox state of the protein, which is in excellent agreement with recent experimental measurements. Assuming the reduced state of heme a(3), His291 of the oxidized Cu(B) center possesses a pK(a)() between 2.1 and 4.0, while His291 of the reduced Cu(B) center has a pK(a) above 17. The obtained results support the proposal that the His291 ligand of the Cu(B) center in CcO is a proton pump element.     

微过氧化物酶水溶液的ABEEM/MM动力学模拟

应用原子-键电负性均衡浮动电荷分子力场(ABEEM/MM), 对微过氧化物酶水溶液进行了分子动力学模拟. 研究了水溶液对微过氧化物酶的结构, 血红素的皱裂构象以及轴配体咪唑基的取向的影响. 结果表明, 在水溶液中微过氧化物酶的骨架氨基酸是稳定的, 而血红素的皱裂构象在水分子的作用下趋于平面. 与血红素轴配体咪唑基键连的组氨酸决定着咪唑基的空间取向, 而咪唑基与血红素侧链的丙酸基的静电作用对其取向仅起次要作用.     

Biomimetic studies of terminal oxidases: trisimidazole picket metalloporphyrins

Three biomimetic models for the binuclear Fe/Cu (heme/trisimidazole) active site of terminal oxidases, such as cytochrome c oxidase and related enzymes, have been prepared. Based upon a tetrakis(aminophenyl)porphyrin core, these models possess a single covalently linked imidazole-bearing tail on one side of the porphyrin and three imidazole "pickets" on the opposite side of the porphyrin ring. Three different imidazole picket motifs are characterized in free base, Fe, Zn, Fe/Cu, and Zn/Cu forms. A combination of NMR, EPR, and IR demonstrates that, for the N-methylimidazole systems studied, the distal Cu is bound within the trisimidazole environment in the reduced (Cu(I)) and oxidized (Cu(II)) forms. The imidazole picket substitution pattern and state of metalation have significant influence on the interaction of these compounds with CO. For imidazole picket systems containing NH groups, intramolecular H bonds compete with Cu(I) coordination of the N donors.     

Ab initio molecular dynamics of heme in cytochrome c

Ab initio molecular dynamics (AIMD) calculations, based on the Car-Parrinello method, have been carried out for three models of heme c that is present in cytochrome c. Both the reduced (Fe(II)) and oxidized (Fe(III)) forms have been analyzed. The simplest models (1R and 1O, respectively) consist of a unsubstituted porphyrin (with no side chains) and two axially coordinated imidazole and ethylmethylthioether ligands. Density functional theory optimizations of these models confirm the basic electronic features and are the starting point for building more complex derivatives. AIMD simulations were performed after reaching the thermal stability at T = 300 K. The evolution of the Fe-L(ax) bond strengths is examined together with the relative rotations of the imidazole and methionine about the axial vector, which appear rather independent from each other. The next models (2R and 2O) contain side chains at the heme to better simulate the actual active site. It is observed that two adjacent propionate groups induce some important effects. The axial Fe-Sdelta bond is only weakened in 2R but is definitely cleaved in the oxidized species 2O. Also the mobility of the Im ligand seems to be reduced by the formation of a strong hydrogen bond that involves the Im Ndelta1-Hdelta1 bond and one carboxylate group. In 2O the interaction becomes so strong that a proton transfer occurs and the propionic acid is formed. Finally, the models 3 include a free N-methyl-acetamide molecule to mimic a portion of the protein backbone. This influences the orientation of carboxylate groups and limits the amount of their hydrogen bonding with the Im ligand. Residual electrostatic interactions are maintained, which are still able to modulate the dissociation of the methionine from the heme.     

Influence of the distal his in imparting imidazolate character to the proximal his in heme peroxidase: (1)h NMR spectroscopic study of cyanide-inhibited his42-->ala horseradish peroxidase

The functional higher oxidation states of heme peroxidases have been proposed to be stabilized by the significant imidazolate character of the proximal His. This is induced by a "push-pull" combination effect produced by the proximal Asp that abstracts ("pulls") the axial His ring N(delta)H, along with the distal protonated His that contributes ("pushes") a strong hydrogen bond to the distal ligand. The molecular and electronic structure of the distal His mutant of cyanide-inhibited horseradish peroxidase, H42A-HRPCN, has been investigated by NMR. This complex is a valid model for the active site hydrogen-bonding network of HRP compound II. The (1)H and (15)N NMR spectral parameters characterize the relative roles of the distal His42 and proximal Asp247 in imparting imidazolate character to the axial His. 1D/2D spectra reveal a heme pocket molecular structure that is highly conserved in the mutant, except for residues in the immediate proximity of the mutation. This conserved structure, together with the observed dipolar shifts of numerous active site residue protons, allowed a quantitative determination of the orientation and anisotropies of the paramagnetic susceptibility tensor, both of which are only minimally perturbed relative to wild-type HRPCN. The quantitated dipolar shifts allowed the factoring of the hyperfine shifts to reveal that the significant changes in hyperfine shifts for the axial His and ligated (15)N-cyanide result primarily from changes in contact shifts that reflect an approximately one-third reduction in the axial His imidazolate character upon abolishing the distal hydrogen-bond to the ligated cyanide. Significant changes in side chain orientation were found for the distal Arg38, whose terminus reorients to partially fill the void left by the substituted His42 side chain. It is concluded that 1D/2D NMR can quantitate both molecular and electronic structural changes in cyanide-inhibited heme peroxidase and that, while both residues contribute, the proximal Asp247 is more important than the distal His42 in imparting imidazole character to the axial His 170.     

Structure of the Sulfoxide Synthase EgtB from the Ergothioneine Biosynthetic Pathway

The non‐heme iron enzyme EgtB catalyzes O₂‐dependent C? S bond formation between γ‐glutamyl cysteine and N‐α‐trimethyl histidine as the central step in ergothioneine biosynthesis. Both, the catalytic activity and the architecture of EgtB are distinct from known sulfur transferases or thiol dioxygenases. The crystal structure of EgtB from Mycobacterium thermoresistibile in complex with γ‐glutamyl cysteine and N‐α‐trimethyl histidine reveals that the two substrates and three histidine residues serve as ligands in an octahedral iron binding site. This active site geometry is consistent with a catalytic mechanism in which C? S bond formation is initiated by an iron(III)‐complexed thiyl radical attacking the imidazole ring of N‐α‐trimethyl histidine.     

How do azoles inhibit cytochrome P450 enzymes? A density functional study

To examine how azole inhibitors interact with the heme active site of the cytochrome P450 enzymes, we have performed a series of density functional theory studies on azole binding. These are the first density functional studies on azole interactions with a heme center and give fundamental insight into how azoles inhibit the catalytic function of P450 enzymes. Since azoles come in many varieties, we tested three typical azole motifs representing a broad range of azole and azole-type inhibitors: methylimidazolate, methyltriazolate, and pyridine. These structural motifs represent typical azoles, such as econazole, fluconazole, and metyrapone. The calculations show that azole binding is a stepwise mechanism whereby first the water molecule from the resting state of P450 is released from the sixth binding site of the heme to create a pentacoordinated active site followed by coordination of the azole nitrogen to the heme iron. This process leads to the breaking of a hydrogen bond between the resting state water molecule and the approaching inhibitor molecule. Although, formally, the water molecule is released in the first step of the reaction mechanism and a pentacoordinated heme is created, this does not lead to an observed spin state crossing. Thus, we show that release of a water molecule from the resting state of P450 enzymes to create a pentacoordinated heme will lead to a doublet to quartet spin state crossing at an Fe-OH(2) distance of approximately 3.0 A, while the azole substitution process takes place at shorter distances. Azoles bind heme with significantly stronger binding energies than a water molecule, so that these inhibitors block the catalytic cycle of the enzyme and prevent oxygen binding and the catalysis of substrate oxidation. Perturbations within the active site (e.g., a polarized environment) have little effect on the relative energies of azole binding. Studies with an extra hydrogen-bonded ethanol molecule in the model, mimicking the active site of the CYP121 P450, show that the resting state and azole binding structures are close in energy, which may lead to chemical equilibrium between the two structures, as indeed observed with recent protein structural studies that have demonstrated two distinct azole binding mechanisms to P450 heme.