Abstract
The contribution of the disulfide bridge in CotA-laccase from Bacillus subtilis is assessed with respect to the enzyme’s functional and structural properties. The removal of the disulfide bond by site-directed mutagenesis, creating the C322A mutant, does not affect the spectroscopic or catalytic properties and, surprisingly, neither the long-term nor the thermodynamic stability parameters of the enzyme. Furthermore, the crystal structure of the C322A mutant indicates that the overall structure is essentially the same as that of the wild type, with only slight alterations evident in the immediate proximity of the mutation. In the mutant enzyme, the loop containing the C322 residue becomes less ordered, suggesting perturbations to the substrate binding pocket. Despite the wild type and the C322A mutant showing similar thermodynamic stability in equilibrium, the holo or apo forms of the mutant unfold at faster rates than the wild-type enzyme. The picosecond to nanosecond time range dynamics of the mutant enzyme was not affected as shown by acrylamide collisional fluorescence quenching analysis. Interestingly, copper uptake or copper release as measured by the stopped-flow technique also occurs more rapidly in the C322A mutant than in the wild-type enzyme. Overall the structural and kinetic data presented here suggest that the disulfide bridge in CotA-laccase contributes to the conformational dynamics of the protein on the microsecond to millisecond timescale, with implications for the rates of copper incorporation into and release from the catalytic centres.
Similar content being viewed by others
References
Lindley PF (2001) In: Bertini I, Sigel A, Sigel H (eds) Handbook on metalloproteins. Dekker, New York, pp 763–811
Messerschmidt A (1997) Multi-copper oxidases. World Science Press, Singapore
Fernandes AT, Damas JM, Todorovic S, Huber R, Baratto MC, Pogni R, Soares CM, Martins LO (2010) FEBS J 277:3176–3189
Fernandes AT, Soares CM, Pereira MM, Huber R, Grass G, Martins LO (2007) FEBS J 274:2683–2694
Martins LO, Soares CM, Pereira MM, Teixeira M, Costa T, Jones GH, Henriques AO (2002) J Biol Chem 277:18849–18859
Durao P, Chen Z, Fernandes AT, Hildebrandt P, Murgida DH, Todorovic S, Pereira MM, Melo EP, Martins LO (2008) J Biol Inorg Chem 13:183–193
Fernandes AT, Martins LO, Melo EP (2009) Biochim Biophys Acta 1794:75–83
Agostinelli E, Cervoni L, Giartosio A, Morpurgo L (1995) Biochem J 306:697–702
Savini I, D’Alessio S, Giartosio A, Morpurgo L, Avigliano L (1990) Eur J Biochem 190:491–495
Sedlak E, Wittung-Stafshede P (2007) Biochemistry 46:9638–9644
Sedlak E, Ziegler L, Kosman DJ, Wittung-Stafshede P (2008) Proc Natl Acad Sci USA 105:19258–19263
Koroleva OV, Stepanova EV, Binukov VI, Timofeev VP, Pfeil W (2001) Biochim Biophys Acta 1547:397–407
Durao P, Bento I, Fernandes AT, Melo EP, Lindley PF, Martins LO (2006) J Biol Inorg Chem 11:514–526
Brockwell DJ (2007) Biochem Soc Trans 35:1564–1568
Pace CN, Hebert EJ, Shaw KL, Schell D, Both V, Krajcikova D, Sevcik J, Wilson KS, Dauter Z, Hartley RW, Grimsley GR (1998) J Mol Biol 279:271–286
Radestock SG H (2008) Eng Life Sci 5:507–522
Zhou XX, Wang YB, Pan YJ, Li WF (2008) Amino Acids 34:25–33
Alcaraz LA, Jimenez B, Moratal JM, Donaire A (2005) Protein Sci 14:1710–1722
Pozdnyakova I, Wittung-Stafshede P (2001) Biochemistry 40:13728–13733
Wittung-Stafshede P (2004) Inorg Chem 43:7926–7933
Bento I, Martins LO, Gato Lopes G, Armenia Carrondo M, Lindley PF (2005) Dalton Trans 3507–3513
Aasa R, Vanngard T (1975) J Magn Reson 19:308–315
Leslie A (1992) CCP4 Newsl Protein Crystallogr 26
Leslie AG (2006) Acta Crystallogr D 62:48–57
Collaborative Computational Project, Number 4 (1994) Acta Crystallogr D 50:760–763
Vagin A, Teplyakov A (1997) J Appl Crystallogr 30:1022–1025
Murshudov GN, Vagin AA, Lebedev A, Wilson KS, Dodson EJ (1999) Acta Crystallogr D 55:247–255
Emsley P, Cowtan K (2004) Acta Crystallogr D Biol Crystallogr 60:2126–2132
Bento I, Silva CS, Chen Z, Martins LO, Lindley PF, Soares CM (2010) BMC Struct Biol 10:28
Brenner AJ, Harris ED (1995) Anal Biochem 226:80–84
Solomon EI, Sundaram UM, Machonkin TE (1996) Chem Rev 96:2563–2606
Moser CC, Dutton PL (1996) In: Bendall DS (ed) Protein electron transfer. Bios Scientific Publishers, Oxford, pp 1–21
Enguita FJ, Martins LO, Henriques AO, Carrondo MA (2003) J Biol Chem 278:19416–19425
Karlin KD, Zhu ZY, Karlin S (1997) Proc Natl Acad Sci USA 94:14225–14230
Durao P, Chen Z, Silva CS, Soares CM, Pereira MM, Todorovic S, Hildebrandt P, Bento I, Lindley PF, Martins LO (2008) Biochem J 412:339–346
Eftink MR, Ghiron CA (1977) Biochemistry 16:5546–5551
Somogyi B, Punyiczki M, Hedstrom J, Norman JA, Prendergast FG, Rosenberg A (1994) Biochim Biophys Acta 1209:61–68
Calhoun DB, Vanderkooi JM, Holtom GR, Englander SW (1986) Proteins 1:109–115
Lakowicz JR (1999) Principles of fluorescence spectroscopy. Kluwer/Plenum, New York
Zhang J, Matthews CR (1998) Biochemistry 37:14891–14899
Leckner J, Bonander N, Wittung-Stafshede P, Malmstrom BG, Karlsson BG (1997) Biochim Biophys Acta 1342:19–27
Pozdnyakova I, Guidry J, Wittung-Stafshede P (2001) Arch Biochem Biophys 390:146–148
Bah A, Garvey LC, Ge J, Di Cera E (2006) J Biol Chem 281:40049–40056
Cawthorn TR, Poulsen BE, Davidson DE, Andrews D, Hill BC (2009) Biochemistry 48:4448–4454
Choi DW, Zea CJ, Do YS, Semrau JD, Antholine WE, Hargrove MS, Pohl NL, Boyd ES, Geesey GG, Hartsel SC, Shafe PH, McEllistrem MT, Kisting CJ, Campbell D, Rao V, de la Mora AM, Dispirito AA (2006) Biochemistry 45:1442–1453
Taniguchi T, Ichimura K, Kawashima S, Yamamura T, Tachi’iri Y, Satake K, Kihara H (1990) Eur Biophys J 18:1–8
Hellman NE, Kono S, Mancini GM, Hoogeboom AJ, De Jong GJ, Gitlin JD (2002) J Biol Chem 277:46632–46638
Blackburn NJ, Ralle M, Hassett R, Kosman DJ (2000) Biochemistry 39:2316–2324
Galli I, Musci G, Bonaccorsi di Patti MC (2004) J Biol Inorg Chem 9:90–95
Kataoka K, Kitagawa R, Inoue M, Naruse D, Sakurai T, Huang HW (2005) Biochemistry 44:7004–7012
Luque I, Leavitt SA, Freire E (2002) Annu Rev Biophys Biomol Struct 31:235–256
Davis-Kaplan SR, Askwith CC, Bengtzen AC, Radisky D, Kaplan J (1998) Proc Natl Acad Sci USA 95:13641–13645
Kwok EY, Severance S, Kosman DJ (2006) Biochemistry 45:6317–6327
Shi X, Stoj C, Romeo A, Kosman DJ, Zhu Z (2003) J Biol Chem 278:50309–50315
Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) J Appl Crystallogr 26:283–291
Acknowledgments
Instituto de Biotecnologia e Química Fina and J.S. Cabral are acknowledged for the use of the Pi-Star 180 instrument for stopped-flow kinetic measurements. The European Synchrotron Radiation Facility in Grenoble, France, and the macromolecular crystallography staff are sincerely acknowledged for provision of synchrotron radiation facilities and support. This work was supported by project grants from Fundação para a Ciência e Tecnologia (FCT), Portugal (POCI/BIO/57083/2004 and PTDC/QUI/73027/2006), and the European Comission (BIORENEW-FP6-2004-NMP-NI-4/026456). A.T.F and C.S.S. hold Ph.D. fellowships (SFRH/BD/31444/2006 and SFRH/BD/40586/2007, respectively) from FCT, Portugal.
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Fernandes, A.T., Pereira, M.M., Silva, C.S. et al. The removal of a disulfide bridge in CotA-laccase changes the slower motion dynamics involved in copper binding but has no effect on the thermodynamic stability. J Biol Inorg Chem 16, 641–651 (2011). https://doi.org/10.1007/s00775-011-0768-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00775-011-0768-9