Dataset Information

The Apparently Unreactive Substrate Facilitates the Electron Transfer for Dioxygen Activation in Rieske Dioxygenases.

ABSTRACT: Rieske dioxygenases belong to the non-heme iron family of oxygenases and catalyze important cis-dihydroxylation as well as O-/N-dealkylation and oxidative cyclization reactions for a wide range of substrates. The lack of substrate coordination at the non-heme ferrous iron center, however, makes it particularly challenging to delineate the role of the substrate for productive O2 activation. Here, we studied the role of the substrate in the key elementary reaction leading to O2 activation from a theoretical perspective by systematically considering (i) the 6-coordinate to 5-coordinate conversion of the non-heme Fe^II upon abstraction of a water ligand, (ii) binding of O2 , and (iii) transfer of an electron from the Rieske cluster. We systematically evaluated the spin-state-dependent reaction energies and structural effects at the active site for all combinations of the three elementary processes in the presence and absence of substrate using naphthalene dioxygenase as a prototypical Rieske dioxygenase. We find that reaction energies for the generation of a coordination vacancy at the non-heme Fe II center through thermoneutral H₂ O reorientation and exothermic O2 binding prior to Rieske cluster oxidation are largely insensitive to the presence of naphthalene and do not lead to formation of any of the known reactive Fe-oxygen species. By contrast, the role of the substrate becomes evident after Rieske cluster oxidation and exclusively for the 6-coordinate non-heme Fe II sites in that the additional electron is found at the substrate instead of at the iron and oxygen atoms. Our results imply an allosteric control of the substrate on Rieske dioxygenase reactivity to happen prior to changes at the non-heme Fe II in agreement with a strategy that avoids unproductive O2 activation.

SUBMITTER: Csizi KS

PROVIDER: S-EPMC9306888 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BACKGROUND:Dihydroxylation of tandemly linked aromatic carbons in a cis-configuration, catalyzed by multicomponent oxygenase systems known as Rieske nonheme iron oxygenase systems (ROs), often constitute the initial step of aerobic degradation pathways for various aromatic compounds. Because such RO reactions inherently govern whether downstream degradation processes occur, novel oxygenation mechanisms involving oxygenase components of ROs (RO-Os) is of great interest. Despite substantial progress in structural and physicochemical analyses, no consensus exists on the chemical steps in the catalytic cycles of ROs. Thus, determining whether conformational changes at the active site of RO-O occur by substrate and/or oxygen binding is important. Carbazole 1,9a-dioxygenase (CARDO), a RO member consists of catalytic terminal oxygenase (CARDO-O), ferredoxin (CARDO-F), and ferredoxin reductase. We have succeeded in determining the crystal structures of oxidized CARDO-O, oxidized CARDO-F, and both oxidized and reduced forms of the CARDO-O: CARDO-F binary complex. RESULTS:In the present study, we determined the crystal structures of the reduced carbazole (CAR)-bound, dioxygen-bound, and both CAR- and dioxygen-bound CARDO-O: CARDO-F binary complex structures at 1.95, 1.85, and 2.00?Å resolution. These structures revealed the conformational changes that occur in the catalytic cycle. Structural comparison between complex structures in each step of the catalytic mechanism provides several implications, such as the order of substrate and dioxygen bindings, the iron-dioxygen species likely being Fe(III)-(hydro)peroxo, and the creation of room for dioxygen binding and the promotion of dioxygen binding in desirable fashion by preceding substrate binding. CONCLUSIONS:The RO catalytic mechanism is proposed as follows: When the Rieske cluster is reduced, substrate binding induces several conformational changes (e.g., movements of the nonheme iron and the ligand residue) that create room for oxygen binding. Dioxygen bound in a side-on fashion onto nonheme iron is activated by reduction to the peroxo state [Fe(III)-(hydro)peroxo]. This state may react directly with the bound substrate, or O-O bond cleavage may occur to generate Fe(V)-oxo-hydroxo species prior to the reaction. After producing a cis-dihydrodiol, the product is released by reducing the nonheme iron. This proposed scheme describes the catalytic cycle of ROs and provides important information for a better understanding of the mechanism.

Project description:The Rieske protein component of the cytochrome bc complex contains a [2Fe-2S] cluster ligated by two cysteines and two histidines. We report here the pK(a) values of each of the imidazole rings of the two ligating histidines (His134 and His154) in the oxidized and reduced states of the Rieske protein from Thermus thermophilus (TtRp) as determined by NMR spectroscopy. Knowledge of these pK(a) values is of critical interest because of their pertinence to the mechanism of electron and proton transfer in the bifurcated Q-cycle. Although we earlier had observed the pH dependence of a (15)N NMR signal from each of the two ligand histidines in oxidized TtRp (Lin, I. J.; Chen, Y.; Fee, J. A.; Song, J.; Westler, W. M.; Markley, J. L. J. Am. Chem. Soc. 2006, 128, 10672-10673), the strong paramagnetism of the [2Fe-2S] cluster prevented the assignment of these signals by conventional methods. Our approach here was to take advantage of the unique histidine-leucine (His134-Leu135) sequence and to use residue-selective labeling to establish a key sequence-specific assignment, which was then extended. Analysis of the pH dependence of assigned (13)C', (13)C(alpha), and (15)N(epsilon2) signals from the two histidine cluster ligands led to unambiguous assignment of the pK(a) values of oxidized and reduced TtRp. The results showed that the pK(a) of His134 changes from 9.1 in oxidized to approximately 12.3 in reduced TtRp, whereas the pK(a) of His154 changes from 7.4 in oxidized to approximately 12.6 in reduced TtRp. This establishes His154, which is close to the quinone when the Rieske protein is in the cytochrome b site, as the residue experiencing the remarkable redox-dependent pK(a) shift. Secondary structural analysis of oxidized and reduced TtRp based upon our extensive chemical shift assignments rules out a large conformational change between the oxidized and reduced states. Therefore, TtRp likely translocates between the cytochrome b and cytochrome c sites by passive diffusion. Our results are most consistent with a mechanism involving the coupled transfer of an electron and transfer of the proton across the hydrogen bond between the hydroquinone and His154 at the cytochrome b site.

Dataset Information

The Apparently Unreactive Substrate Facilitates the Electron Transfer for Dioxygen Activation in Rieske Dioxygenases.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets