Tognaccini L.,University of Florence |
Ciaccio C.,University of Rome Tor Vergata |
Ciaccio C.,Interuniversity Consortium for the Research |
D'Oria V.,University of Rome Tor Vergata |
And 9 more authors.
Journal of Inorganic Biochemistry | Year: 2016
Spectroscopic and functional properties of human cytochrome c and its Tyr67 residue mutants (i.e., Tyr67His and Tyr67Arg) have been investigated. In the case of the Tyr67His mutant, we have observed only a very limited structural alteration of the heme pocket and of the Ω-loop involving, among others, the residue Met80 and its bond with the heme iron. Conversely, in the Tyr67Arg mutant the Fe-Met80 bond is cleaved; consequently, a much more extensive structural alteration of the Ω-loop can be envisaged. The structural, and thus the functional modifications, of the Tyr67Arg mutant are present in both the ferric [Fe(III)] and the ferrous [Fe(II)] forms, indicating that the structural changes are independent of the heme iron oxidation state, depending instead on the type of substituting residue. Furthermore, a significant peroxidase activity is evident for the Tyr67Arg mutant, highlighting the role of Arg as a basic, positively charged residue at pH 7.0, located in the heme distal pocket, which may act as an acid to cleave the O-O bond in H2O2. As a whole, our results indicate that a delicate equilibrium is associated with the spatial arrangement of the Ω-loop. Clearly, Arg, but not His, is able to stabilize and polarize the negative charge on the Fe(III)-OOH complex during the formation of Compound I, with important consequences on cytochrome peroxidation activity and its role in the apoptotic process, which is somewhat different in yeast and mammals. © 2015 Elsevier Inc.
Gioia M.,University of Rome Tor Vergata |
Gioia M.,Interuniversity Consortium for the Research |
Tsukada H.,University of Tennessee Health Science Center |
Liang J.,University of Tennessee Health Science Center |
And 7 more authors.
Journal of Biological Inorganic Chemistry | Year: 2012
The role of the hinge region in the unwinding and cleavage of type I collagen by interstitial collagenase (MMP-1) has been studied at 37 C and pH 7.3. The collagenolytic processing by MMP-1 displays a very similar overall rate for both chains of collagen I, even though the affinity is higher for the a-1 chain and the cleavage rate is faster for the a-2 chain. MMP-1 binding to collagen I brings about a significant unwinding of the triple-helical arrangement only after the first cleavage step of the a-1 and a-2 chains. The proteolytic processing by wild-type MMP- 1 on a synthetic substrate and collagen I has been compared with that observed for site-directed mutants obtained either by truncating the hinge region (D255-272) or by individually replacing the conserved amino acids Val268, Gly272, and Lys277 of the hinge region with residues observed for the corresponding position in stromelysin-1 (MMP-3), a noncollagenolytic metalloproteinase. The D256-272 mutant has no collagenolytic activity, clearly demonstrating the crucial role of this region for the enzymatic processing of collagen I. However, among various mutants investigated, only Gly272Asp shows a dramatically reduced enzymatic activity both on the synthetic substrate and on collagen I. This effect, however, is clearly related to the substituting residue, since substitution of Ala or Asn for Gly272 does not have any effect on the kinetic properties of MMP-1. These data suggest that the substrate specificity of MMP-1 is dictated by the reciprocal structural relationships between the catalytic domain and the carboxyterminal domain through the conformational arrangement of the hinge region. © SBIC 2012.