Single mutation induces a metal-dependent subunit association in dimeric Cu,Zn superoxide dismutase

Tryptophan 83, a residue strongly involved in the intersubunit interaction of the Cu,Zn superoxide dismutases from Photobacterium leiognathi, has been selectively mutated to phenylalanine or tyrosine. The recombinant mutant enzymes expressed in Escherichia coil were purified in two well distinct and stable forms, one dimeric and fully active and the other monomeric and devoid of metals. In agreement, in vitro experiments indicate that the removal and addition of zinc in the mutant enzymes induces monomerization and dimerization, respectively, while does not perturb the dimeric association of the native protein. This is the first unambiguous experimental proof of a direct communication between the intersubunit interface and the metal active site. (C) 2000 Academic Press

LOW-TEMPERATURE OPTICAL SPECTROSCOPY OF NATIVE AND AZIDE-REACTED BOVINE CU,ZN SUPEROXIDE-DISMUTASE - A STRUCTURAL DYNAMICS STUDY RID F-2353-2010 RID F-2664-2011 RID A-4573-2009

The optical absorption spectra of native and N-3(-)-reacted Cu,Zn superoxide dismutase (SOD) has been studied in the temperature range 300-10 K. The broad d-d bands observed in the room temperature spectrum, centered at 14 700 cm(-1) (native enzyme) and at 15 550 cm(-1) (N-3(-)-reacted enzyme), are clearly split at low temperature into two bands each, centered at 12 835 and 14 844 cm(-1) and at 14 418 and 16 300 cm(-1), respectively. The thermal behavior of the 23 720 cm(-1) band present in the spectrum of the native enzyme indicates that this band belongs to the His61-->Cu(IT) ligand to metal charge transfer transition. Analysis of the zeroth, first, and second moments of the various bands as a function of temperature allowed us to obtain useful information on the stereodynamic properties of the metal site in SOD. In particular for the native protein, it was possible to infer a variation in the metal ligand relative position that occurs as the temperature is lowered and that likely involves all of the ligands except His61. On the other hand, the site is stabilized upon N-3(-) binding, and in this case a variation in the metal ligand position is observed only at the level of the bound anion. The possible relation of these properties to the catalytic mechanism of the enzyme is discussed

Low-temperature optical spectroscopy of cobalt in Cu,Co superoxide dismutase: A structural dynamics study of the solvent-unaccessible metal site RID F-2353-2010 RID F-2664-2011 RID A-4573-2009

The temperature dependence (300 to 10 K) of the electronic absorption spectra of the cobalt chromophore in bovine superoxide dismutase (SOD) having the native Zn(II) ion selectively replaced by Co(II) has been investigated in four different derivatives: Cu(II),Co(II) SOD, N-3(-)-Cu(II),Co(II) SOD, Cu(I),Co(II) SOD, and E,Co(II) SOD in which the copper ion has been selectively removed. In the Cu(II),Co(lI) SOD, the cobalt spectrum is characterized at room temperature by three bands centered at 18,472, 17,670, and 16,793 cm(-1); the low-frequency band is split, at low temperatures, into two components, indicating a lower symmetry contribution to a predominantly tetrahedral crystal field. Addition of N-3(-) to the Cu(II),Co(II) SOD introduces slight changes in all the Co(II) visible bands, indicating the occurrence of minor perturbations of the structural cobalt site upon anion binding to the catalytic copper site. Analysis of the spectra in the Cu(I),Co(II) and E,Co(II) enzymes indicates that the His61 imidazolate bridge is released from the copper upon reduction. This is also confirmed by the analysis of the zeroth, first, and second moments of the various bands in the different derivatives. The cobalt site is characterized by a harmonic dynamics, at variance with what observed in the solvent accessible copper site [Cupane, A., Leone, M., Militello, V., Stroppolo, M. E., Polticelli, F., & Desideri, A. (1994) Biochemistry 33, 15103-15109]. The degree of local microheterogeneity at the cobalt site is smaller than that observed for the copper site and increases in the order N-3(-)-Cu(II),Co(II)approximate to Cu(I),Co(II) < Cu(I),Co(II) < E,Co(II) indicating a different local packing and the presence of different constraints on the cobalt site in the four derivatives. The different dynamic behavior with respect to the catalytic, solvent-accessible, copper site is discussed

Single mutation at the intersubunit interface confers extra efficiency to Cu,Zn superoxide dismutase.

The Val28--&gt;Gly single mutant at the subunit interface of Cu,Zn superoxide dismutase from Photobacterium leiognathi displays a k(cat)/K(M) value of 1.7x10(10) M(-1) s(-1), twice that of the native enzyme. Analysis of the three-dimensional structure indicates that the active site Cu,Zn center is not perturbed, slight structural deviations being only localized in proximity of the mutation site. The enzyme-substrate association rate, calculated by Brownian dynamics simulation, is identical for both enzymes, indicating that the higher catalytic efficiency of the Val28--&gt;Gly mutant is not due to a more favorable electrostatic potential distribution. This result demonstrates the occurrence of an intramolecular communication between the mutation site and the catalytic center, about 18 A away and indicates a new strategy to encode extra efficiency within other members of this enzymatic family