Model Complexes Elucidate the Role of the Proximal Hydrogen-Bonding Network in Cytochrome P450s

Abstract

Cytochrome (Cyt) P450s are an important class of enzymes with numerous functions in nature. The unique reactivity of these enzymes relates to their heme b active sites with an axially bound, deprotonated cysteine (a “cysteinate”) ligand (chemically speaking a thiolate). The heme-thiolate active sites further contain a number of conserved hydrogen-bonds (H-bonds) to the bound cysteinate ligand, which have been proposed to tune and stabilize the Fe–S bond. In this work, we present the low-temperature preparation of five ferric heme-thiolate nitric oxide (NO) model complexes that contain one tunable hydrogen-bond to the bound thiolate ligand. We show that the presence of a H-bond has a dramatic effect in stabilizing the thiolate ligand against direct reaction with NO. This observation reinforces the important protective role of H-bonds in Cyt P450s. We further demonstrate that H-bond strength tunes thiolate donor strength, which, in turn, controls the N–O and Fe–NO stretching frequencies and hence, bond strengths. We observe a direct correlation between the Fe–NO and N–O stretching frequencies, indicative of a thiolate σ-trans effect (interaction). Here, very small changes in H-bond strength lead to a surprisingly large effect on the FeNO unit. This result implies that subtle changes in the Cys-pocket of a Cyt P450 can strongly affect reactivity. Importantly, using the Fe–NO/N–O correlation established here, the thiolate donor strength in heme-thiolate enzyme active sites and model complexes can be quantified in a straightforward way, using NO as a probe. This spectroscopic correlation provides a quantitative measure of the thiolate’s “push” effect, which is important in O2-activation (Compound I formation) in Cyt P450s in general.

The Biologically Relevant Coordination Chemistry of Iron and Nitric Oxide: Electronic Structure and Reactivity

“…P450 (heme-thiolate) enzyme mimics, , and the collective experimental results available for the NO adducts of different heme-thiolate enzymes. ,− With findings (i)–(iv) now firmly established, based on experimental data, it is concluded that the differences in Fe–NO and N–O stretching frequencies between different heme-thiolate ls-{FeNO} 6 protein complexes are largely due to variations in the number and strength of hydrogen bonds to their respective cysteinate sulfurs. Figure compares the data points of the 11 heme-thiolate ls-{FeNO} 6 model complexes with those reported for ls-{FeNO} 6 adducts in a number of heme-thiolate proteins (see also Table ), and shows the two correlation lines established in the model complex studies. , The slopes obtained from two different linear fits of the data points in Figure (1.20 and 1.61) establish a direct correlation of the Fe–NO and N–O stretching frequencies, and hence bond strengths, with the donor strength of the axial thiolate ligand. For example, moving from right to left in the correlation plot, the thiolate donor strength increases, leading to a simultaneous weakening of both the Fe–NO and N–O bonds.…”

“…These complexes were obtained by the reaction of the corresponding 5C ferric heme-thiolate precursors (all with TPP 2– as the porphyrin ligand) with NO at −80 °C, and subsequently characterized using low-temperature UV–vis, IR, and rRaman spectroscopy ( Figure ). In these model systems, thiophenolate ligands were used that either contain (a) various electron-withdrawing substituents (i.e., the “electron poor” thiolate series), or (b) one intramolecular hydrogen bond to the thiolate sulfur (ligands – SPh-NHPh- p R, where R is a functional group allowing for tunability of the hydrogen-bond strength), , as shown in Scheme . These model systems allowed for the experimental demonstration that in heme-thiolate ls-{FeNO} 6 complexes: Both electron-withdrawing groups and hydrogen bonds can modulate the thiolate donor strength in a similar way. ,, The thiolate donor strength directly modulates the Fe–NO and N–O bond strengths via a thermodynamic σ- trans effect (more precisely, a σ- trans interaction, since it is a thermodynamic effect) that can be experimentally quantified by spectroscopic determination of the Fe–NO and N–O stretching frequencies. Hydrogen bonds are able to provide additional protection for the thiolate ligand against S -nitrosylation and potentially other side reactions as well. ,, The cumulative strength of the proximal hydrogen bonds to the thiolate ligand in proteins and model systems can in turn be gauged by determination of the Fe–NO and N–O stretching frequencies of the corresponding ls-{FeNO} 6 adducts, by comparison of these data to the vibrational correlation plot shown in Figure . …”

“…(B) Low-temperature (−80 °C) UV–vis spectra monitoring the formation of the ls-{FeNO} 6 heme thiolate complex [Fe­(TPP)­(SPh-NHPh- p CH 3 )­(NO)] from the reaction of the corresponding 5C ferric heme-thiolate complex with NO gas. Adapted with permission from ref . Copyright 2020 American Chemical Society.…”

“…(B) The thiophenolate ligand series that contains one intramolecular hydrogen bond (indicated by a dashed line). Here, the R group can be varied to tune the strength of this hydrogen bond. , .…”

“…Abbreviations: CPO = chloroperoxidase. Adapted with permission from ref . Copyright 2020 American Chemical Society.…”