Perturbations of the T1 copper site in the CotA laccase from Bacillus subtilis: structural, biochemical, enzymatic and stability studies

Abstract

Site-directed mutagenesis has been used to replace Met502 in CotA laccase by the residues leucine and phenylalanine. X-ray structural comparison of M502L and M502F mutants with the wild-type CotA shows that the geometry of the T1 copper site is maintained as well as the overall fold of the proteins. The replacement of the weak so-called axial ligand of the T1 site leads to an increase in the redox potential by approximately 100 mV relative to that of the wild-type enzyme (E ⁰=455 mV). However the M502L mutant exhibits a twofold to fourfold decrease in the k _cat values for the all substrates tested and the catalytic activity in M502F is even more severely compromised; 10% activity and 0.15–0.05% for the non-phenolic substrates and for the phenolic substrates tested when compared with the wild-type enzyme. T1 copper depletion is a key event in the inactivation and thus it is a determinant of the thermodynamic stability of wild-type and mutant proteins. Whilst the unfolding of the tertiary structure in the wild-type enzyme is a two-state process displaying a midpoint at a guanidinium hydrochloride concentration of 4.6 M and a free-energy exchange in water of 10 kcal/mol, the unfolding for both mutant enzymes is clearly not a two-state process. At 1.9 M guanidinium hydrochloride, half of the molecules are in an intermediate conformation, only slightly less stable than the native state (approximately 1.4 kcal/mol). The T1 copper centre clearly plays a key role, from the structural, catalytic and stability viewpoints, in the regulation of CotA laccase activity.

Appendix

The thermodynamic stability of CotA wild type monitored by fluorescence was analysed according to a two-state process (N↔U) using the following equations:

$$ y = y_{{\text{N}}} f_{{\text{N}}} + y_{{\text{U}}} f_{{\text{U}}} , $$

(1)

$$ K_{{({\text{U}} - {\text{N}})}} = f_{{\text{U}}} /f_{{\text{N}}} , $$

(2)

$$ \Delta G^{{\text{0}}}_{{({\text{U}} - {\text{N}})}} = - RT\,\ln \,K_{{({\text{U}} - {\text{N}})}} , $$

(3)

$$ \Delta G^{{\text{0}}}_{{({\text{U}} - {\text{N}})}} = \Delta G^{{0\,{\text{ water}}}}_{{({\text{U}} - {\text{N}})}} - m_{{({\text{U}} - {\text{N}})}} [{\text{GdnHCl}}] $$

(4)

and

$$ [{\text{GdnHCl}}]_{{50\% }} = \Delta G^{{0\,{\text{ water}}}}_{{({\text{U}} - {\text{N}})}} /m_{{({\text{U}} - {\text{N}})}} , $$

(5)

where N and U are native and unfolded CotA, respectively, y is the fluorescence signal, f is the fraction of CotA molecules with a given conformation, K is the equilibrium constant, ΔG ⁰ is the standard free energy, m _(U–N) is the linear dependence of ΔG ⁰ on GdnHCl concentration and [GdnHCl]_50% is the GdnHCl concentration for ΔG ⁰=0. y _N and y _U were calculated directly from the pretransition and posttransition regions according to a linear dependence.

The thermodynamic stability evaluated by activity measurements and the absorbance at 600 nm are dependent on copper loss from T1. Therefore Eqs. 1, 2, 3, 4 and 5 were used but considering that the two-state process assessed by these two techniques is the establishment between the native state with copper bound at T1 and the native state with no copper at T1 (N↔N_{(no copper)}).

The thermodynamic stability of CotA M502L and M502F mutants monitored by fluorescence could only be accurately fitted according to a three-state process, with the accumulation of an intermediate state in-between N and U (N↔I↔U). The following equations were used:

$$ y = y_{{\text{N}}} f_{{\text{N}}} + y_{{\text{I}}} f_{{\text{I}}} + y_{{\text{U}}} f_{{\text{U}}} , $$

(6)

$$ K_{{({\text{I}} - {\text{N}})}} = f_{{\text{I}}} /f_{{\text{N}}} , $$

(7)

$$ K_{{({\text{U}} - {\text{I}})}} = f_{{\text{U}}} /f_{{\text{I}}} , $$

(8)

$$ \Delta G^{{\text{0}}}_{{({\text{I}} - {\text{N}})}} = - RT\,\ln \,K_{{({\text{I}} - {\text{N}})}} , $$

(9)

$$ \Delta G^{{\text{0}}}_{{({\text{U}} - {\text{I}})}} = - RT\,\ln \,K_{{({\text{U}} - {\text{I}})}} , $$

(10)

$$ \Delta G^{{\text{0}}}_{{({\text{I}} - {\text{N}})}} = \Delta G^{{0\,{\text{ water}}}}_{{({\text{I}} - {\text{N}})}} - m_{{({\text{I}} - {\text{N}})}} [{\text{GdnHCl}}] $$

(11)

and

$$ \Delta G^{{\text{0}}}_{{({\text{U}} - {\text{I}})}} = \Delta G^{{0\,{\text{ water}}}}_{{({\text{U}} - {\text{I}})}} - m_{{({\text{U}} - {\text{I}})}} [{\text{GdnHCl}}], $$

(12)

where y _N was considered to be the fluorescence signal at 0 M GdnHCl (this assumption was confirmed by the fluorescence emission maximum) and y _U was calculated directly from the posttransition regions according to a linear dependence.

Combining Eqs. 6, 7, 8, 9, 10, 11 and 12, we fitted the fluorescence signal (and therefore fractional change of fluorescence signal) according to Eq. 13:

$$ y = \frac{\begin{aligned} & \left( {y_{\text{N}} + y_{\text{I}} \exp \left( {\left( { - \Delta G_{({\text{I}} - {\text{N}})} ^{0{\text{water}}} + m_{({\text{I}} - {\text{N}})} \left[ {{\text{GdnHCl}}} \right]} \right)/RT} \right)} \right. \\ & \quad \left. { + y_{\text{U}} \exp \left( {\left( { - \Delta G_{({\text{I}} - {\text{N}})} ^{0{\text{water}}} + m_{({\text{I}} - {\text{N}})} \left[ {{\text{GdnHCl}}} \right]} \right)/RT} \right)\exp \left( {\left( { - \Delta G_{({\text{U}} - {\text{I}})} ^{0{\text{water}}} + m_{({\text{U}} - {\text{I}})} \left[ {{\text{GdnHCl}}} \right]} \right)/RT} \right)} \right) \\ \end{aligned} } {\begin{aligned} & \left( {\exp \left( {\left( { - \Delta G_{({\text{I}} - {\text{N}})} ^{0{\text{water}}} + m_{({\text{I}} - {\text{N}})} \left[ {{\text{GdnHCl}}} \right]} \right)/RT} \right)} \right. \\ & \quad \left. { + 1 + \exp \left( {\left( { - \Delta G_{({\text{I}} - {\text{N}})} ^{0{\text{water}}} + m_{({\text{I}} - {\text{N}})} \left[ {{\text{GdnHCl}}} \right]} \right)/RT} \right)\exp \left( {\left( { - \Delta G_{({\text{U}} - {\text{I}})} ^{0{\text{water}}} + m_{({\text{U}} - {\text{I}})} \left[ {{\text{GdnHCl}}} \right]} \right)/RT} \right)} \right) \\ \end{aligned} }. $$

(13)

The fits were carried out by varying values of y _I, ΔG ^0 water_(I–N) , m _(I–N), ΔG ^0 water_(U–I) and m _(U–I) with the Origin software using the non-linear curve-fit option. Combination of Eqs. 6, 7 and 8 leads to

$$ f_{{\text{I}}} = \frac{{K_{{({\text{I}} - {\text{N}})}} }} {{1 + K_{{({\text{I}} - {\text{N}})}} + K_{{({\text{I}} - {\text{N}})}} K_{{({\text{U}} - {\text{I}})}} }}$$

(14)

and

$$ f_{{\text{U}}} = \frac{{K_{{({\text{I}} - {\text{N}})}} K_{{({\text{U}} - {\text{I}})}} }} {{1 + K_{{({\text{I}} - {\text{N}})}} + K_{{({\text{I}} - {\text{N}})}} K_{{({\text{U}} - {\text{I}})}} }}.$$

(15)