Structural insights into the role of the acid-alcohol pair of residues required for dioxygen activation in cytochrome P450 enzymes

The cytochrome P450 heme monooxygenases commonly use an acid-alcohol pair of residues, within the I-helix, to activate iron-bound dioxygen. This work aims to clarify conflicting reports on the importance of the alcohol functionality in this process. Mutants of the P450, CYP199A4 (CYP199A4D251N and CYP199A4T252A), were prepared, characterised and their crystal structures were solved. The acid residue of CYP199A4 is not part of a salt bridge network, a key feature of paradigmatic model system P450cam. Instead, there is a direct proton delivery network, via a chain of water molecules, extending to the surface. Nevertheless, CYP199A4D251N dramatically reduced the activity of the enzyme consistent with a role in proton delivery. CYP199A4T252A decreased the coupling efficiency of the enzyme with a concomitant increase in the hydrogen peroxide uncoupling pathway. However, the effect of this mutation was much less pronounced than reported with P450cam. Its crystal structures revealed fewer changes at the I-helix, compared to the P450cam system. The structural changes observed within the I-helix of P450cam during oxygen activation do not seem to be required in this P450. These differences are due to the presence of a second threonine residue at position 253, which is absent in P450cam. This threonine forms part of the hydrogen bonding network, resulting in subtle structural changes and is also present across the majority of the P450 superfamily. Overall, the results suggest that while the acid-alcohol pair is important for dioxygen activation this process and the method of proton delivery can differ across P450s.Tom Coleman, Jeanette E. Stok, Matthew N. Podgorski, John B. Bruning, James J. De Voss, Stephen G. Bel

Biophysical techniques for distinguishing ligand binding modes in cytochrome P450 monooxygenases

Published: February 14, 2020The cytochrome P450 superfamily of heme monooxygenases catalyzes important chemical reactions across nature. The changes in the optical spectra of these enzymes, induced by the addition of substrates or inhibitors, are critical for assessing how these molecules bind to the P450, enhancing or inhibiting the catalytic cycle. Here we use the bacterial CYP199A4 enzyme (Uniprot entry Q2IUO2 ), from Rhodopseudomonas palustris HaA2, and a range of substituted benzoic acids to investigate different binding modes. 4-Methoxybenzoic acid elicits an archetypal type I spectral response due to a ≥95% switch from the low- to high-spin state with concomitant dissociation of the sixth aqua ligand. 4-(Pyridin-3-yl)- and 4-(pyridin-2-yl)benzoic acid induced different type II ultraviolet-visible (UV-vis) spectral responses in CYP199A4. The former induced a greater red shift in the Soret wavelength (424 nm vs 422 nm) along with a larger overall absorbance change and other differences in the α-, β-, and δ-bands. There were also variations in the ferrous UV-vis spectra of these two substrate-bound forms with a spectrum indicative of Fe-N bond formation with 4-(pyridin-3-yl)benzoic acid. The crystal structures of CYP199A4, with the pyridinyl compounds bound, revealed that while the nitrogen of 4-(pyridin-3-yl)benzoic acid is coordinated to the heme, with 4-(pyridin-2-yl)benzoic acid an aqua ligand remains. Continuous wave and pulse electron paramagnetic resonance data in frozen solution revealed that the substrates are bound in the active site in a form consistent with the crystal structures. The redox potential of each CYP199A4-substrate combination was measured, allowing correlation among binding modes, spectroscopic properties, and the observed biochemical activity.Matthew N. Podgorski, Joshua S. Harbort, Tom Coleman, Jeanette E. Stok, Jake A. Yorke, Luet-Lok Wong, John B. Bruning, Paul V. Bernhardt, James J. De Voss, Jeffrey R. Harmer, and Stephen G. Bel

The transformation of post-communist economies in a globalised economy : the case of Poland

This article argues that the transformation of the economies of Central and Eastern Europe (CEE) has to be understood in the context of the dynamics and development of the global economy. The analysis draws on the notion of combined and uneven development in which there has recently been renewed interest. Too often this notion has been a slogan that lacks substance, but the article elaborates how change is a dynamic process of interaction between economic change and political and social forces. The neoliberal analysis, as well as some Marxist accounts, are criticised for being deterministic, linear and prescriptive. This account emphasises the institutional dimension and role of the state as being critical to understanding the varied outcomes between and within economies in CEE in terms of the way that it has mediated the reinsertion of these countries into the global economy. The story focuses on agency, a neglected aspect of analysis, in emphasising the ideological and discursive aspects of transformation, which attempt to justify and reinforce economic and material changes and to close down debate about alternatives. Crucially, the form and content of development, in its widest sense, cannot be known or predicted because the process of transformation has been contested by different factions of the ruling class and by workers. Despite the marginalisation of organised labour in mainstream and many radical accounts, it is argued that trade unions and workers have been central to the process and outcomes of transformationNon peer reviewe