Role of substrate on the conformational stability of the heme active site of cytochrome P450cam: effect of temperature and low concentrations of denaturants

Abstract

The effect of 1R-camphor on the conformational stability of the heme active site of cytochrome P450cam has been investigated. The absorption spectra of the heme moiety showed the presence of two hitherto unknown intermediates formed at low urea concentrations or during small temperature perturbations. The corresponding thermodynamic parameters were obtained by global fitting of the experimental data to a generalized sequential unfolding model at different wavelengths, which showed that the active conformation of the enzyme is stabilized by binding of the substrate at the active site. Circular-dichroism spectra of the enzyme in the visible- and far-UV region were studied to identify the critical range of denaturant concentration and the temperature at which the tertiary structure around the heme center was affected with almost no change in the secondary structure of the enzyme. This critical range of urea concentration was 0–2.8 M in the presence of camphor and 0–1.5 M in the absence of camphor. The tertiary structure of the enzyme was found to undergo conformational change in the temperature range 20–60 °C in the presence of the substrate and 20–47 °C in its absence. The spectral assignments of the intermediate species of the heme active site with the intact secondary structure of the enzyme were made by deconvolution of the Soret absorption spectra, and the results were analyzed to determine stabilization of the heme active-site geometry by 1R-camphor. Results showed that subtle conformational changes due to melting of the tertiary contacts in the active site lead to formation of intermediates which are coordinatively similar to the native enzyme. Analogous intermediate species might be responsible for leakage in the redox catalytic cycle of the enzyme.

Appendix

Let us assume a general unfolding model with n sequential (reversible) intermediates. The scheme can be written as follows:

$$ N \rightleftarrows I_{1} \rightleftarrows I_{2} \rightleftarrows I_{3} \rightleftarrows I_{4} \rightleftarrows \ldots \rightleftarrows I_{{n - 1}} \rightleftarrows I_{n} \rightleftarrows U{\left( { \approx I_{{n + 1}} } \right)} $$

$$ \begin{array}{*{20}c} {{K_{1} = {I_{1} } \mathord{\left/ {\vphantom {{I_{1} } N}} \right. \kern-\nulldelimiterspace} N = {k_{1} } \mathord{\left/ {\vphantom {{k_{1} } {k_{{ - 1}} }}} \right. \kern-\nulldelimiterspace} {k_{{ - 1}} }}} \\ {{K_{{n + 1}} = U \mathord{\left/ {\vphantom {U {I_{n} }}} \right. \kern-\nulldelimiterspace} {I_{n} } = {k_{{n + 1}} } \mathord{\left/ {\vphantom {{k_{{n + 1}} } {k_{{ - {\left( {n + 1} \right)}}} }}} \right. \kern-\nulldelimiterspace} {k_{{ - {\left( {n + 1} \right)}}} }}} \\ {{K_{i} = {I_{i} } \mathord{\left/ {\vphantom {{I_{i} } {I_{{i - 1}} }}} \right. \kern-\nulldelimiterspace} {I_{{i - 1}} } = {k_{1} } \mathord{\left/ {\vphantom {{k_{1} } {k_{{ - i}} }}} \right. \kern-\nulldelimiterspace} {k_{{ - i}} }}} \\ \end{array} $$

(3)

where N denotes native protein, U denotes unfolded form, I is concentration of the respective intermediates, k values are corresponding rate constants, and K _i (mol⁻¹) is the equilibrium constant for the ith step. The coupled linear equation for the aforementioned scheme can be written as:

$$ k_{1} N_{0} - k_{1} {\sum\nolimits_{i = 1}^{n + 1} {I_{i} - k_{{ - 1}} } } \times I_{1} = 0 $$

(4)

where $I_{i} = {\left( {{k_{i} } \mathord{\left/ {\vphantom {{k_{i} } {k_{{ - i}} }}} \right. \kern-\nulldelimiterspace} {k_{{ - i}} }} \right)} \times I_{{i - 1}} = K_{i} I_{{i - 1}}$ and ${\sum\nolimits_{i = 1}^{n + 1} {I_{i} = } }{\left( {{I_{1} } \mathord{\left/ {\vphantom {{I_{1} } {K_{1} }}} \right. \kern-\nulldelimiterspace} {K_{1} }} \right)} \times {\sum\nolimits_{i = 1}^{n + 1} {{\left\lfloor {\prod ^{i}_{{j = 1}} K_{j} } \right\rfloor }} }$.

Therefore:

$$ I_{i} = \frac{{K_{1} \times N_{0} }} {{1 + {\sum\nolimits_{i = 1}^{n + 1} {{\left\{ {\prod ^{i}_{{j = 1}} K_{j} } \right\}}} }}} $$

(5)

Any spectroscopic variable (absorbance, circular dichroism or fluorescence) at given wavelength can be expressed as:

$$ A^{\lambda }_{{{\text{obs}}}} = \varepsilon ^{\lambda }_{N} {\left( {N_{0} - {\sum\nolimits_{i = 1}^{n + 1} {\varepsilon ^{\lambda }_{{I_{i} }} I_{i} } }} \right)} + {\sum\nolimits_{i = 1}^{n + 1} {\varepsilon ^{\lambda }_{{I_{i} }} I_{i} } } $$

(6)

Now using Eqs. (4) and (5), Eq. (6) can be rewritten as:

$$ A^{\lambda }_{{{\text{obs}}}} = A^{\lambda }_{0} - N_{0} \times \frac{{{\sum\nolimits_{i = 1}^{n + 1} {{\left\{ {{\left( {\varepsilon ^{\lambda }_{N} - \varepsilon ^{\lambda }_{{I_{i} }} } \right)} \times {\left\{ {\prod ^{i}_{{j = 1}} K_{j} } \right\}}} \right\}}} }}} {{1 + {\sum\nolimits_{i = 1}^{n + 1} {{\left\{ {\prod ^{i}_{{j = 1}} K_{j} } \right\}}} }}} $$

Using (from linear energy-model assumption):

$$ \Delta G^{j} = \Delta G^{j}_{0} - m_{j} {\left[ D \right]} $$

(7)

$$ A^{\lambda }_{{{\text{obs}}}} {\left[ D \right]} = A^{\lambda }_{0} - N_{0} \times \frac{{{\sum\nolimits_{i = 1}^{n + 1} {{\left\{ {{\left( {\varepsilon ^{\lambda }_{N} - \varepsilon ^{\lambda }_{{I_{i} }} } \right)} \times \prod ^{i}_{{j = 1}} e^{{{ - {\left( {\Delta G^{j}_{0} - m_{j} D} \right)}} \mathord{\left/ {\vphantom {{ - {\left( {\Delta G^{j}_{0} - m_{j} D} \right)}} {RT}}} \right. \kern-\nulldelimiterspace} {RT}}} } \right\}}} }}} {{1 + {\sum\nolimits_{i = 1}^{n + 1} {{\left\{ {\prod ^{i}_{{j = 1}} e^{{{ - {\left( {\Delta G^{j}_{0} - m_{j} D} \right)}} \mathord{\left/ {\vphantom {{ - {\left( {\Delta G^{j}_{0} - m_{j} D} \right)}} {RT}}} \right. \kern-\nulldelimiterspace} {RT}}} } \right\}}} }}} $$

(8)

Using the following approximation:

$$ \Delta G^{{{\text{H}}_{{\text{2}}} {\text{O}}}}_{{T_{i} }} \cong \Delta H_{{m_{i} }} - T\Delta S_{{m_{i} }} $$

(9)

$$ A^{\lambda }_{{{\text{obs}}}} {\left[ T \right]} = A^{\lambda }_{0} - N_{0} \times \frac{{{\sum\nolimits_{i = 1}^{n + 1} {{\left\{ {{\left( {\varepsilon ^{\lambda }_{N} - \varepsilon ^{\lambda }_{{I_{i} }} } \right)} \times \prod ^{i}_{{j = 1}} e^{{{ - \Delta G^{{{\text{H}}_{{\text{2}}} {\text{O}}}}_{{Tj}} } \mathord{\left/ {\vphantom {{ - \Delta G^{{{\text{H}}_{{\text{2}}} {\text{O}}}}_{{Tj}} } {RT}}} \right. \kern-\nulldelimiterspace} {RT}}} } \right\}}} }}} {{1 + {\sum\nolimits_{i = 1}^{n + 1} {{\left\{ {\prod ^{i}_{{j = 1}} e^{{{ - \Delta G^{{{\text{H}}_{{\text{2}}} {\text{O}}}}_{{Tj}} } \mathord{\left/ {\vphantom {{ - \Delta G^{{{\text{H}}_{{\text{2}}} {\text{O}}}}_{{Tj}} } {RT}}} \right. \kern-\nulldelimiterspace} {RT}}} } \right\}}} }}} $$

(10)

Equations (8) and (10) are used in Eqs. (1) and (2) of Materials and method. An intermediate I _i is said to be spectroscopically silent at a particular wavelength λ, when $<!!!>{\left( {\varepsilon ^{\lambda }_{N} - \varepsilon ^{\lambda }_{{I_{i} }} } \right)} = 0$. In this case, though the corresponding components in Eqs. (8) and (10) become zero, the total free energy is an invariant, i.e., the free-energy component of the ith intermediate will be added to the free energy of the (i+1)th intermediate thus making the total free energy a constant.