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Oxidative Modification of Aldose Reductase Induced by Copper Ion

2002; Elsevier BV; Volume: 277; Issue: 44 Linguagem: Inglês

10.1074/jbc.m206945200

1083-351X

I Cecconi, Andrea Scaloni, Giulio Rastelli, Maria Moroni, Pier Giuseppe Vilardo, Luca Costantino, Mario Cappiello, Donita Garland, Deborah Carper, J. Mark Petrash, Antonella Del Corso, Umberto Mura,

Heme Oxygenase-1 and Carbon Monoxide

Aldose reductase (ALR2) is susceptible to oxidative inactivation by copper ion. The mechanism underlying the reversible modification of ALR2 was studied by mass spectrometry, circular dichroism, and molecular modeling approaches on the enzyme purified from bovine lens and on wild type and mutant recombinant forms of the human placental and rat lens ALR2. Two equivalents of copper ion were required to inactivate ALR2: one remained weakly bound to the oxidized protein whereas the other was strongly retained by the inactive enzyme. Cys303 appeared to be the essential residue for enzyme inactivation, because the human C303S mutant was the only enzyme form tested that was not inactivated by copper treatment. The final products of human and bovine ALR2 oxidation contained the intramolecular disulfide bond Cys298-Cys303. However, a Cys80-Cys303 disulfide could also be formed. Evidence for an intramolecular rearrangement of the Cys80-Cys303 disulfide to the more stable product Cys298-Cys303 is provided. Molecular modeling of the holoenzyme supports the observed copper sequestration as well as the generation of the Cys80-Cys303disulfide. However, no evidence of conditions favoring the formation of the Cys298-Cys303 disulfide was observed. Our proposal is that the generation of the Cys298-Cys303 disulfide, either directly or by rearrangement of the Cys80-Cys303 disulfide, may be induced by the release of the cofactor from ALR2 undergoing oxidation. The occurrence of a less interactive site for the cofactor would also provide the rationale for the lack of activity of the disulfide enzyme forms. Aldose reductase (ALR2) is susceptible to oxidative inactivation by copper ion. The mechanism underlying the reversible modification of ALR2 was studied by mass spectrometry, circular dichroism, and molecular modeling approaches on the enzyme purified from bovine lens and on wild type and mutant recombinant forms of the human placental and rat lens ALR2. Two equivalents of copper ion were required to inactivate ALR2: one remained weakly bound to the oxidized protein whereas the other was strongly retained by the inactive enzyme. Cys303 appeared to be the essential residue for enzyme inactivation, because the human C303S mutant was the only enzyme form tested that was not inactivated by copper treatment. The final products of human and bovine ALR2 oxidation contained the intramolecular disulfide bond Cys298-Cys303. However, a Cys80-Cys303 disulfide could also be formed. Evidence for an intramolecular rearrangement of the Cys80-Cys303 disulfide to the more stable product Cys298-Cys303 is provided. Molecular modeling of the holoenzyme supports the observed copper sequestration as well as the generation of the Cys80-Cys303disulfide. However, no evidence of conditions favoring the formation of the Cys298-Cys303 disulfide was observed. Our proposal is that the generation of the Cys298-Cys303 disulfide, either directly or by rearrangement of the Cys80-Cys303 disulfide, may be induced by the release of the cofactor from ALR2 undergoing oxidation. The occurrence of a less interactive site for the cofactor would also provide the rationale for the lack of activity of the disulfide enzyme forms. aldose reductase bovine lens ALR2 human placental recombinant ALR2 h-C80S, h-C303S, cysteine to serine mutated h-ALR2 rat lens recombinant ALR2 cysteine to serine mutated r-ALR2 carboxamidomethyl dithiothreitol glutathione-modified ALR2 liquid chromatography-electrospray ionization mass spectrometry 2-mercaptoethanol molecular dynamics molecular mechanics Transition metals have a relevant role among systems that can induce or modulate oxidative stress. They are both able to promote the formation of reactive oxygen species (1Halliwell B. Gutteridge M.C. Biochem. J. 1984; 219: 1-14Crossref PubMed Scopus (4503) Google Scholar) and to act as cofactors in enzymatic systems devoted to counteract oxidative stress. The important role of the copper ion as an effective prosthetic group for special protein functions and its role as a potential toxic agent in cell function are handled by the cell through a fine control of the free copper level by highly efficient metal chelating proteins (2Stückel J. Wallace A.C. Cohen F.E. Prusiner S.B. Biochemistry. 1998; 37: 7185-7193Crossref PubMed Scopus (490) Google Scholar, 3Linder M.C. Hazegh-Azam M. Am. J. Clin. Nutr. 1996; 63: 797S-811SPubMed Google Scholar). In this regard, it is worth noting the extensive cell damage associated with pathologies resulting from both excess and a deficit of copper, such as Wilson's and Menkes's disease, respectively (4Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (388) Google Scholar, 5Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (527) Google Scholar). When the concentration of free copper increases, either by environmental or pathological causes, cell damage likely occurs (6Ogihara H. Ogihara T. Miki M. Yasuda H. Mino M. Pediatr. Res. 1995; 37: 219-226Crossref PubMed Scopus (112) Google Scholar, 7Tallis G.A. Kitchener M.I. Thomas A.C. Clin. Chem. 1990; 36: 568-570Crossref PubMed Scopus (10) Google Scholar, 8Harman D. J. Gerontol. 1965; 20: 151-153Crossref PubMed Scopus (126) Google Scholar, 9Brewer G.J. Yuzbasiyan-Gurkan V. Medicine. 1992; 71: 139-164Crossref PubMed Scopus (351) Google Scholar, 10Lin J. Jpn. J. Ophthalmol. 1977; 41: 130-137Crossref Scopus (28) Google Scholar). The effectiveness of copper ion in inducing protein as well as nucleic acid oxidation, by eliciting the generation of reactive oxygen species through a Fenton-type reaction, is well documented (11Ueda J. Saito N. Ozawa T. Arch. Biochem. Biophys. 1996; 325: 65-76Crossref PubMed Scopus (17) Google Scholar, 12Li Y. Trush M.A. Yager J.D. Carcinogenesis. 1994; 15: 1421-1427Crossref PubMed Scopus (147) Google Scholar, 13Kobayashi S. Ueda K. Morita J. Sakai H. Komano T. Biochim. Biophys. Acta. 1988; 949: 143-147Crossref PubMed Scopus (30) Google Scholar, 14Gutteridge J.M. Halliwell B. Biochem. Pharmacol. 1982; 31: 2801-2805Crossref PubMed Scopus (109) Google Scholar, 15Aruoma O.I. Halliwell B. Gajewski E. Dizdaroglu M. Biochem. J. 1991; 273: 601-604Crossref PubMed Scopus (439) Google Scholar, 16Garland D. Exp. Eye Res. 1990; 50: 677-682Crossref PubMed Scopus (111) Google Scholar). Moreover, because of its ability to bind proteins and nucleic acids, copper has the potential to specifically promote in situoxidative modification reactions. Thus, it is important to define the mechanisms underlying processes induced by copper-protein interaction. The effect of the copper ion on aldose reductase (alditol:NADP+ oxidoreductase, EC 1.1.1.21) (ALR2),1 isolated from bovine lens was previously described (17Cecconi I. Moroni M. Vilardo P.G. Dal Monte M. Borella P. Rastelli G. Costantino L. Garland D. Carper D. Petrash J.M. Del Corso A. Mura U. Biochemistry. 1998; 37: 14167-14174Crossref PubMed Scopus (18) Google Scholar). It appears that the enzyme, which was previously shown to be especially susceptible to thiol-mediated oxidation (18Giannessi M. Del Corso A. Cappiello M. Voltarelli M. Marini I. Barsacchi D. Garland D. Camici M. Mura U. Arch. Biochem. Biophys. 1993; 300: 423-429Crossref PubMed Scopus (31) Google Scholar, 19Cappiello M. Voltarelli M. Cecconi I. Vilardo P.G. Dal Monte M. Marini I. Del Corso A. Wilson D.K. Quiocho F.A. Petrash J.M. Mura U. J. Biol. Chem. 1996; 271: 33539-33544Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 20Liu S.-Q. Bhatnagar A. Srivastava S.K. Biochim. Biophys. Acta. 1992; 1120: 329-366Crossref PubMed Scopus (25) Google Scholar, 21Cappiello M. Voltarelli M. Giannessi M. Cecconi I. Camici G. Manao G. Del Corso A. Mura U. Exp. Eye Res. 1994; 58: 491-501Crossref PubMed Scopus (58) Google Scholar, 22Vilardo P.G. Scaloni A. Amodeo P. Barsotti C. Cecconi I. Cappiello M. Lopez Mendez B. Rullo R. Dal Monte M. Del Corso A. Mura U. Biochemistry. 2001; 40: 11985-11994Crossref PubMed Scopus (17) Google Scholar), is highly sensitive to Cu(II). The enzyme is readily inactivated by the metal ion through an oxygen independent modification process. The modified enzyme, fully reactivated in the presence of dithiothreitol, was postulated to contain an intramolecular disulfide bond and to carry two equivalents of bound copper ion. Based on the characterization of the inactivation process and on the measurement of the redox state of the bound copper on the enzyme, it was concluded that the metal ion responsible for ALR2 inactivation was directly involved in a site specific oxidation mechanism of the enzyme. In this paper, the rationale for the definition of the copper binding site(s) and the formation of the disulfide bond in ALR2 is put forward through mass spectrometry, circular dichroism, and molecular modeling approaches on the enzyme and its mutants from different species. NADPH, d,l-glyceraldehyde, dithiothreitol, GSH, EDTA, endoproteinase Lys-C, iodoacetamide, DTT, and myoglobin were purchased from Sigma. Bathocuproinedisulfonic acid was from Jannsen Pharmaceutical. All electrophoresis reagents and isoelectric focusing standards were from Bio-Rad. Ampholine PAG plates, pH 4.0–6.5, for isoelectric focusing were from Amersham Biosciences. Copper(II) chloride and all inorganic chemicals were of reagent grade and were from BDH. The ALR2 inhibitor (S)(+)-6-fluoro-2,3-dihydrospiro[4H-1-benzopyran-4,4′-imidazolidine]-2′,5′-dione (Sorbinil) (23Inagaki K. Miwa I. Yashiro T. Okuda J. Chem. Pharm. Bull. 1982; 30: 3244-3254Crossref PubMed Scopus (47) Google Scholar) was a gift from Dr. G. Caccia, Laboratori Baldacci S.p.A., Pisa, Italy. The complex (bathocuproinedisulfonic acid)2Cu(I) was a gift from Dr. R. L. Levine, Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, MD. γ-Glutamyl-cysteinyl-2-[3H]glycine ([3H]GSH), 1 Ci/mol was purchased from PerkinElmer Life Sciences. The purification of b-ALR2 was performed as previously described (24Del Corso A. Barsacchi D. Giannessi M. Tozzi M.G. Camici M. Houben J.L. Zandomeneghi M. Mura U. Arch. Biochem. Biophys. 1990; 283: 512-518Crossref PubMed Scopus (41) Google Scholar). The pure native enzyme (specific activity 1.12 units/mg) was stored at 4 °C in 10 mm sodium phosphate buffer, pH 7.0 (S-buffer), supplemented with 2 mm DTT. Expression of h-ALR2 in Escherichia coli was done as previously described (25Petrash J.M. Harter T.M. Devine C.S. Olins P.O. Bhatnagar A. Liu S. Srivastava S.K. J. Biol. Chem. 1992; 267: 24833-24840Abstract Full Text PDF PubMed Google Scholar). Recombinant aldose reductase was extracted from host cells by osmotic shock and stored at −70 °C until used. Wild type and mutated forms of h-ALR2 were purified to electrophoretic homogeneity by the same chromatographic steps used for the bovine lens enzyme (24Del Corso A. Barsacchi D. Giannessi M. Tozzi M.G. Camici M. Houben J.L. Zandomeneghi M. Mura U. Arch. Biochem. Biophys. 1990; 283: 512-518Crossref PubMed Scopus (41) Google Scholar); the pure enzymes were stored at 4 °C in S-buffer supplemented with 2 mm DTT. The specific activities of h-ALR2 and its C298S, C80S, and C303S mutants were 3.9, 9.8, 3.1, and 6.2 units/mg, respectively. E. coli expressing r-ALR2 and its mutants was grown as previously described (26Old S.E. Sato S. Kador P.F. Carper D.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4942-4945Crossref PubMed Scopus (23) Google Scholar). The cells were washed twice in 20 mm imidazole buffer, pH 7.2, and centrifuged. The cell pellet was resuspended in 10 ml of the same buffer, sonicated, and stored at −70 °C until used. r-ALR2 and its mutants were purified to electrophoretic homogeneity by the same chromatographic steps used for the bovine lens enzyme (24Del Corso A. Barsacchi D. Giannessi M. Tozzi M.G. Camici M. Houben J.L. Zandomeneghi M. Mura U. Arch. Biochem. Biophys. 1990; 283: 512-518Crossref PubMed Scopus (41) Google Scholar); the pure enzymes were stored at 4 °C in S-buffer supplemented with 2 mm DTT. The specific activities of r-ALR2 and its H200Q, H110Q, H41Q, H187Q, and C298S mutants were 4.5, 5.2, 0.6, 4.0, 4.0, and 5.2 units/mg, respectively. The ALR2 activity and sensitivity to inhibition by Sorbinil were measured as previously described by usingd,l-glyceraldehyde as substrate (17Cecconi I. Moroni M. Vilardo P.G. Dal Monte M. Borella P. Rastelli G. Costantino L. Garland D. Carper D. Petrash J.M. Del Corso A. Mura U. Biochemistry. 1998; 37: 14167-14174Crossref PubMed Scopus (18) Google Scholar). Before use, the enzyme forms were extensively dialyzed against S-buffer. If not otherwise specified, copper treatment of h-ALR2 forms was performed by supplementing the enzyme after dialysis with a stoichiometric amount of NADP+. When r-ALR2 was used, 30 μm DTT was present in the dialysis buffer and 4 μm DTT was constantly present in all further incubations. All tested enzyme forms, from 3 to 8 μm final concentrations, were incubated for the proper time at 25 °C in S-buffer supplemented with CuCl2 to give final ratios of [Cu(II)]/[enzyme] from 0.5 to 5, as specifically indicated. At the end of the incubation, 0.5 mm EDTA was added and the enzyme activity was measured. To detect copper bound to ALR2, the samples were extensively dialyzed at 4 °C against S-buffer containing 0.5 mm EDTA. The concentration of Cu(I) was determined by a complexometric method as previously described (17Cecconi I. Moroni M. Vilardo P.G. Dal Monte M. Borella P. Rastelli G. Costantino L. Garland D. Carper D. Petrash J.M. Del Corso A. Mura U. Biochemistry. 1998; 37: 14167-14174Crossref PubMed Scopus (18) Google Scholar) by measuring the formation of the complex between the metal ion and bathocuproinedisulfonic acid. Circular dichroism spectra were obtained on a Jasco J40AS spectropolarimeter with a cylindrical 10-mm path length cuvette kept at 10 °C. A spectral bandwidth of 2 nm was used. To block reduced cysteines, ALR2 samples were alkylated with 1.1m iodoacetamide in 0.25 m Tris-HCl, 1.25 mm EDTA, containing 6 m guanidinium chloride, pH 7.0, at room temperature for 1 min in the dark. Proteins were freed from salt and reagent excess by passing the reaction mixture through an analytical Vydac C4 column as previously reported (22Vilardo P.G. Scaloni A. Amodeo P. Barsotti C. Cecconi I. Cappiello M. Lopez Mendez B. Rullo R. Dal Monte M. Del Corso A. Mura U. Biochemistry. 2001; 40: 11985-11994Crossref PubMed Scopus (17) Google Scholar). Protein samples were manually collected and lyophilized. Electrospray mass spectra of intact protein species were recorded by using an API-100 single quadrupole mass spectrometer (Applied Biosystems) equipped with an atmospheric pressure ionization source as previously reported (22Vilardo P.G. Scaloni A. Amodeo P. Barsotti C. Cecconi I. Cappiello M. Lopez Mendez B. Rullo R. Dal Monte M. Del Corso A. Mura U. Biochemistry. 2001; 40: 11985-11994Crossref PubMed Scopus (17) Google Scholar). Mass calibration was performed by means of the multiply charged ions from a separate injection of horse heart myoglobin (molecular mass 16,951.5 Da). All masses are reported as average values. Samples of carboxamidomethylated aldose reductase (150 μg) were digested with endoproteinase Lys-C in 0.4% ammonium bicarbonate, pH 8.0, at 37 °C overnight, using an enzyme/substrate ratio of 1:100 (w/w). ALR2 digests were analyzed using a LCQ Deca mass spectrometer (ThermoFinnigan) equipped with an electrospray source connected to a HP1100 chromatographic system (Agilent, Palo Alto, CA). Peptide mixtures were separated on a narrow bore Vydac C18 column (The Separation Group) using a linear gradient from 5 to 70% acetonitrile containing 0.1% trifluoroacetic acid, over a period of 65 min, at a flow rate of 0.2 ml/min. The column effluent was split 1:1 into the mass spectrometer connected on-line. The remaining part was spectrophotometrically detected at 220 nm. In the last case, peptides were manually collected and lyophilized for further characterization. Spectra were acquired in the range m/z 250–2000. Data were elaborated using the Excalibur software provided by the manufacturer. The instrument was calibrated using a mixture of caffeine, MRFA peptide, and Ultramark 1621. A determination of the relative abundance of the peptides containing Cys80, Cys298, and Cys303 was performed as previously reported by Vinci et al. (27Vinci F. Ruoppolo M. Pucci P. Freedman R.B. Marino G. Protein Sci. 2000; 9: 525-535Crossref PubMed Scopus (26) Google Scholar). Briefly, because different peptides containing a specific Cys residue could exist, the ion current for peptides containing a specific Cys residue (in reduced or oxidized form) was obtained by summing the ion current relative to all peptides containing that cysteine (in reduced or oxidized form). To obtain the relative abundance of a specific cysteine in reduced or oxidized form, this value was divided by the ion current produced by all peptides containing Cys80, Cys298, and Cys303. Because different peptides may ionize with different efficiencies, it was not possible to evaluate the absolute abundance of each reduced or oxidized cysteine. It was, nevertheless, possible to compare the trends in the oxidation of the different cysteine residues. Automated N-terminal degradation of the purified peptides was performed by using Procise 491 protein sequencer (Applied Biosystems) equipped with a 140C microgradient apparatus and a 785A UV detector (Applied Biosystems) for the automated identification of phenylthiohydantoin-derivative. Protein concentration was determined according to Bradford (28Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211946) Google Scholar) using bovine serum albumin as the standard. Electrophoretic and isoelectrofocusing analyses were performed as previously described (17Cecconi I. Moroni M. Vilardo P.G. Dal Monte M. Borella P. Rastelli G. Costantino L. Garland D. Carper D. Petrash J.M. Del Corso A. Mura U. Biochemistry. 1998; 37: 14167-14174Crossref PubMed Scopus (18) Google Scholar). Radioactivity measurements were done using a Beckman LS5000CE scintillation counter and Optiphase Hi Safe as scintillation fluid with a counting efficiency of 50% as determined by the tritium standard quench curve of the instrument. Molecular mechanics and molecular dynamics simulations were performed with the sander_classic module of AMBER6 (29Case D.A. Pearlman D.A. Caldwell J.W. Cheathan III, T.E. Ross W.S. Simmerling C.L. Darden T.A. Merz K.M. Stanton R.V. Cheng A.L. Vincent J.J. Crowley M. Tsui V. Radmer R.J. Duan Y. Pitera J. Massova I. Seibel G.L. Singh U.C. Weiner P.K. Kollman P.A. AMBER6. University of California, San Francisco, CA1999Google Scholar), using the Cornell et al. (30Cornell W.D. Cieplak P. Bayly C.I. Gould I.R. Merz K.M. Ferguson D.M. Spellmeyer D.C. Fox T. Caldwell J.W. Kollman P.A. J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11373) Google Scholar) force field. Calculations were performed on a IBM-SP3 computer. Graphical display and manipulations were performed on Silicon Graphics O2 workstations using MIDAS (31Ferrin T.E. Huang C.C. Jarvis L.E. Langridge L. J. Mol. Graph. 1988; 6: 13-27Crossref Scopus (927) Google Scholar). Force field parameters for copper(II) have been developed in the present work and tested on plastocyanin. An ionic (nonbonded) approach (32Hay B.P. Coord. Chem. Rev. 1993; 126: 177-236Crossref Scopus (190) Google Scholar) was adopted in modeling the metal. The van der Waals R* of copper(II) was interpolated from a linear plot of the R* values of the metal ions contained in the AMBER force field against their atomic radii (33Weast R.C. Astle M.J. Beyer W.H. Handbook of Chemistry and Physics. 66th Edition. CRC Press, Boca Raton, FL1985–1986Google Scholar); similarly, the well depth ε was interpolated from an exponential plot of the ε values of the same metal ionsversus their atomic radii. Values of R* = 1.26 Å and ε = 0.0123 kcal/mol were obtained. The coordination geometry of copper extracted from the crystal structure of plastocyanin (34Guss J.M. Bartunik H.D. Freeman H.C. Acta Cryst. Sect. B. 1992; 48: 790-811Crossref PubMed Scopus (280) Google Scholar) was used for charge calculations of copper; to this aim, the Cα carbons of the four amino acids coordinating copper (His37, His87, Cys84, and Met92) were truncated with methyl groups, and cysteine was modeled as anionic (35Ryde U. Ollson M.H.M. Pierloot K. Roos B.O. J. Mol. Biol. 1996; 261: 586-596Crossref PubMed Scopus (159) Google Scholar, 36Pierloot K., De Kerpel J.O.A. Ryde U. Roos B.O. J. Am. Chem. Soc. 1997; 119: 218-226Crossref Scopus (110) Google Scholar). The atomic charge of Cu(II) was obtained from an electrostatic potential fit to STO-3G and 6–31G* ab initio wave functions, using GAUSSIAN94 (37Frisch M.J. Trucks G.W. Schlegel H.B. Gill M.W. Johnson B.G. Robb M.A. Cheeseman J.R. Keith T. Petersson G.A. Montgomery J.A. Raghavachari K., Al- Laham M.A. Zakrzewski V.G. Ortiz J.V. Gaussian94, Revision D. Gaussian, Inc., Pittsburgh, PA1995Google Scholar), followed by standard RESP fit (38Bayly C.I. Cieplak P. Cornell W.D. Kollman P.A. J. Phys. Chem. 1993; 97: 10269-10277Crossref Scopus (5352) Google Scholar, 39Cieplak P. Bayly C.I. Cornell W.D. Kollman P.A. J. Comput. Chem. 1995; 16: 1357-1377Crossref Scopus (845) Google Scholar). In the first case, a STO-3G basis set for copper and the whole coordination sphere was used, whereas a mixed 6–31G* basis set for the amino acids and a STO-3G basis set for copper was used in a second case. Atomic charges of copper of +0.71 and +1.13 were obtained, respectively. In both cases, the formal +2 charge of copper turned out to be significantly reduced upon coordination. As plastocyanin might not be an appropriate model for the copper coordination in ALR2, which is unknown, charge calculations were repeated for a system comprised of copper and six coordinating water molecules, using STO-3G. We still found that the charge of copper reduces to +1.08, a finding that confirms that the charge of the metal has to be reduced from +2 in liquid simulations, regardless of the nature of the residues that coordinate the metal. To test the reliability of the present R*, ε, and charge parameters of copper, MM and MD calculations were performed on the whole plastocyanin molecule using AMBER. Hydrogens were added to the protein and then optimized, keeping the heavy atoms of the protein fixed at their original positions. Counterions were placed around the charged residues at the surface of the protein to neutralize the system. The parameters for Na+ and Cl− were taken from the works of Åqvist (40Åqvist J. J. Phys. Chem. 1990; 94: 8021-8024Crossref Scopus (1406) Google Scholar) and Jorgensen (41Jorgensen W.L. Buckner J.K. Huston S.E. Rossky P.J. J. Am. Chem. Soc. 1987; 109: 1891-1899Crossref Scopus (133) Google Scholar), respectively. Crystallographic water molecules buried inside the protein were maintained and a 15-Å spherical cap of TIP3P (42Jorgensen W.L. Chandrasekhar J. Madura J.D. Impey R.W. Klein M.L. J. Chem. Phys. 1983; 79: 926-935Crossref Scopus (28658) Google Scholar) water molecules centered on copper was added, resulting in 83 additional waters. Three independent MM and MD simulations were performed by setting the charge on copper at +2 (formal charge), +1.13 (mixed 6–31G*/STO-3G), and +0.71 (ST0–3G). In each case, 3,000 steps of conjugate-gradient minimization with MM were performed on the water molecules first, followed by 10,000 steps minimization of the protein residues at 12-Å distance from copper and all the water molecules. A 10-Å cut-off for the nonbonded interactions was adopted. Molecular dynamics was performed for 100 ps at 27 °C, using SHAKE (43van Gusteren W.F. Berendsen H.J.C. Mol. Phys. 1977; 34: 1311-1327Crossref Scopus (1560) Google Scholar) to constrain bond lengths at their equilibrium values. Coordinates were collected every 0.1 ps for analysis, with the last 20 ps averaged for analysis. The root mean square deviations between the averaged structures and the crystal structure of plastocyanin (34Guss J.M. Bartunik H.D. Freeman H.C. Acta Cryst. Sect. B. 1992; 48: 790-811Crossref PubMed Scopus (280) Google Scholar) have been analyzed. Taking into account the root mean square deviation values corresponding to the backbone atoms of the protein, values of 0.50, 0.36, and 0.28 were obtained for the simulations employing the +2, +1.13, and +0.71 charge on copper, respectively. Root mean square deviations limited to the coordination sphere of copper (i.e. copper, His37, His87, Cys84, and Met92) gave a similar trend (0.47, 0.28, and 0.20 for the three simulations). Therefore, MD with the formal charge of +2 on copper gave the worst results both in terms of reproducing the crystal structure of plastocyanin and the coordination geometry of copper. In contrast, the +0.71 simulation gave the best results and this charge was used throughout for the aldose reductase simulations. As it will be shown, an ad hoc strategy was devised to simulate the modification of ALR2 as a two-step process in which copper initially forms a noncovalent complex with ALR2 and, subsequently, induces the disulfide bridge formation. Two copper ions were docked into the structure of the human ALR2 holoenzyme (44Wilson D.K. Bohren K.M. Gabbay K.H. Quiocho F.H. Science. 1992; 257: 81-84Crossref PubMed Scopus (388) Google Scholar). Because Cys80, Cys298, and Cys303 are the three cysteines involved in the formation of a disulfide bridge, copper ions were initially positioned to interact with these residues. One copper ion was positioned close to Cys80 and one close to Cys303 to investigate the formation of the Cys80-Cys303 disulfide, and one copper ion was positioned close to Cys298 and one close to Cys303 for the Cys298-Cys303 disulfide. When coordinating copper, cysteines were assigned a deprotonated form (35Ryde U. Ollson M.H.M. Pierloot K. Roos B.O. J. Mol. Biol. 1996; 261: 586-596Crossref PubMed Scopus (159) Google Scholar, 36Pierloot K., De Kerpel J.O.A. Ryde U. Roos B.O. J. Am. Chem. Soc. 1997; 119: 218-226Crossref Scopus (110) Google Scholar). The ALR2 structures have been prepared using a procedure similar to that described for plastocyanin, with hydrogens added and counterions placed. The parameters for the cofactor were taken from previous work. Structures were solvated with spherical caps of more than 2000 TIP3P (42Jorgensen W.L. Chandrasekhar J. Madura J.D. Impey R.W. Klein M.L. J. Chem. Phys. 1983; 79: 926-935Crossref Scopus (28658) Google Scholar) water molecules centered on the center of mass of ALR2. The following protocol was adopted for minimization and dynamics. A few steps of minimization with MM were performed on the two copper ions keeping the protein fixed at its original position to adjust their initial position with respect to the two cysteines. Prior to energy minimization of ALR2, only the water molecules were energy minimized and then subjected to 50 ps of MD at 27 °C to let the solvent equilibrate around the solute. Then, 5000 steps of minimization were performed on the whole system. 300 ps of MD at 27 °C was then performed starting from the minimized structure, using the same conditions described for plastocyanin. The whole structure was allowed to move during MD. MD was continued for over 800 ps in the case of the enzyme loaded with the two copper ions close to Cys298 and Cys303. Because the sulfur atoms of Cys80 and Cys303, after the 300 ps MD with copper, turned out to be much closer than the corresponding atoms in the crystal structure of the holoenzyme, the last minimized structure obtained from the noncovalent simulation described in the force field parameter section was used as the starting point for building a covalent disulfide bond between these cysteines. Five thousand steps of minimization and 800 ps of MD at 27 °C were performed on the enzyme carrying the Cys80-Cys303 disulfide with coordinates collected for the subsequent analysis. Because, after 800 ps of MD, Cys298 and Cys303were not sufficiently close to form a disulfide in the noncovalent complexes, a different strategy was used to build a model structure carrying this disulfide. Using the homology modeling software Model er6 (45Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10292) Google Scholar), the sequence of ALR2 was artificially aligned with itself, with the only difference being that while the template did not contain any disulfide (the crystal structure of the holoenzyme), a disulfide was explicitly requested in the modeled structure. This approach produced an initial structure of ALR2 carrying the disulfide; the quality of the structure was evaluated using PROCHECK (46Laskowski R.A. McArthur M.W. Moss D.S. Thornton J.M. J. Appl. Cryst. 1993; 26: 283-291Crossref Google Scholar). Then, the two copper ions were docked close to Cys298 and Cys303, and the structure was refined with MM and MD using the same protocol described above. 5000 steps of minimization were performed, and the structure was equilibrated with 900 ps of MD. The recombinant h-ALR2 and r-ALR2 were readily inactivated by low concentrations of Cu(II) (Fig.1). The rate and extent of inactivation were dependent on copper ion concentration. Moreover, in both cases enzyme activity was recovered upon addition of DTT. Following incubation with CuCl2 and extensive dialysis at 4 °C against EDTA, r-ALR2 and h-ALR2 contained 1.8 ± 0.1 and 2.1 ± 0.1 equivalents of total metal ion per enzyme mole, respectively. This is consistent with that reported for the b-ALR2 (17Cecconi I. Moroni M. Vilardo P.G. Dal Monte M. Borella P. Rastelli G. Costantino L. Garland D. Carper D. Petrash J.M. Del Corso A. Mura U. Biochemistry. 1998; 37: 14167-