Why is CarH photolytically active in comparison to other B12-dependent enzymes?
Graphical abstract
Introduction
While the light sensitivity of the cobalt‑carbon (Co-C5′) bond in coenzyme B12 (AdoCbl = adenosylcobalamin, Fig. 1) has been known for nearly five decades, only until recently has this been associated with light controlled reactivity, namely optogenetic regulation and light-activated drug delivery [1,2]. In particular, the CarH photoreceptor, uses AdoCbl as a photoactive cofactor capable of inducing significant structural changes of the photoreceptor's protein chains to regulate DNA transcription [[3], [4], [5]]. Coenzyme B12 contains a Co(III) ion that is equatorially coordinated to four nitrogens of a corrin ring and perpendicularly to upper and lower ligands. The upper ligand is variable and used to distinguish Cbls from one another in B12 nomenclature [6]. In CarH, the upper axial ligand is a 5′-deoxy-5'adenosyl (Ado, Fig. 1) and is covalently bound to the Co via the C5′ of the ribose moiety. The photolytic scission of the Co-C5′ bond of AdoCbl is a key feature of CarH's mechanism of action [7,8]. In B12-dependent enzymes, the lower ligand is the dimethylbenzimidazole (DBI) base from the nucleotide loop or a histidine (His) residue [6,9,10]. In the CarH photoreceptor, the DBI base is replaced with His177, forming the base-off/His-on conformation of the cofactor [5].
The CarH photoreceptor regulates DNA transcription in bacteria which results in the expression of genes responsible for biosynthesis of carotenoids [[3], [4], [5]]. This involves significant structural changes to the photoreceptor as well as local, molecular level processes in the Cbl binding domain. The overall mode of action for CarH is depicted in Fig. 2. In the dark, CarH-DS (DS = Dark State), forms a tetramer upon AdoCbl binding. Transcription of carotenoid biosynthetic genes is repressed in the absence of daylight, where CarH-DS binds to DNA. In the presence of light, the Co-C5′ bond is ruptured, and the Ado ligand is converted to the photoproduct, 4′,5′-anhydroadenosine [7]. As a result, His132 will take the place of the Ado ligand, forming a stable adduct with the cofactor [4]. This photoinitiated process induces a large scale structural change in the protein's conformation and the tetramer dissociates into monomers forming CarH-LS (LS = Light State). In CarH-LS, the cofactor retains His132 and His177 as upper and lower axial ligands, respectively, and this is denoted as bis-HisCbl [5]. Ultimately, this cascade of events initiates the transcription of carotenoid biosynthetic genes.
Although the ‘big picture’ mode of action for CarH seems to be generally understood, there are many issues and questions about the photo-catalytic mechanism that have not been explored to date. One important question has arisen: “how is the Co-C5′ bond activated and cleaved in CarH-DS?” This seemingly simple question has in fact been quite perplexing because AdoCbl is known for its radical chemistry and, in terms of photochemistry, the cleavage of the Co-C5′ bond of AdoCbl is homolytic [6,9,[11], [12], [13], [14], [15]]. It came as quite a surprise that a proposed mechanism for CarH, implicated heterolytic cleavage of the Co-C5′ bond over homolytic cleavage [8].
Even more intriguing is a second question: “what specifically makes CarH photolytically active in comparison to other AdoCbl-dependent enzymes?” Photolytic properties of AdoCbl-dependent enzymes, including glutamate mutase (GLM) and ethanolamine ammonia-lyase (EAL), have been thoroughly investigated both experimentally [[16], [17], [18], [19], [20]] and computationally [[21], [22], [23]]. It was found that photo-cleavage of the Co-C5′ in GLM and EAL enzymes is suppressed and only a small portion of the Co-C5′ bonds undergoes photodissociation. In stark contrast to CarH, excited state dynamics and photolytic studies of GLM and EAL have revealed that these enzymes were essentially photostable. Intuitively, it is reasonable to suppose that there must be some aspect at the molecular level that is responsible for the different photo-reactivities of these enzymes especially considering that coenzyme B12 is utilized in each system mentioned above.
The purpose of this study is to answer these two questions. Quantum mechanics/molecular mechanics (QM/MM) calculations were employed to probe the photolytic properties of CarH-DS and to determine what structural features of the AdoCbl cofactor contribute to these properties. When comparing the excited state properties, exemplified by the S1 state potential energy surface (PES), of CarH to the previously studied GLM and EAL, there are not any major differences. However, based on theoretical insights, it is apparent that the conformation of the Ado group of the cofactor significantly contributes to CarH's efficiency as a photoreceptor. Present calculations do not indicate that the protein environment in the CarH active center alters the photochemistry of AdoCbl, rather it appears that CarH alters the stereochemistry of the ribose moiety to allow for its photolytic activity. This will undoubtedly have implications for elucidating the entire CarH mechanism at the molecular level and in analyses of other similar photoreceptors.
Section snippets
Activation and Photolytic Co-C5′ Bond Cleavage
To understand how CarH operates at the molecular level it is paramount to analyze the PESs associated with the ground state (S0) and the lowest electronically excited state (S1). The S1 state is particularly important because photocleavage of the Co-C5′ bond and the subsequent radical pair (RP) formation occurs from here [1]. DFT/MM and TD-DFT/MM were employed for generating the S0 and S1 PESs, respectively. The S0 and S1 PESs were constructed as a function of axial bond lengths, Co-C5′ and Co-N
Conclusions
Present QM/MM calculations clearly indicate that role of light in the mechanism of CarH-DS is simply to generate the Co(II)/Ado• RP, in a similar fashion that has been observed for EAL and GLM . This is evident based on the similar topologies of the S1 PESs of CarH, EAL, and GLM. In addition, unlike some B12-dependent enzymes like MetH, an energetically feasible route for photodissociation can be identified on the S1 PES of CarH-DS. It is also apparent that AdoCbl plays a unique dual role in
Computational Methods
The CarH model used in the QM/MM calculations was generated from the crystal structure of Thermus thermophilus CarH-DS bound to DNA (PDB ID: 5C8E) [4]. The computational details for GLM and EAL are included in the SI. Additionally, SI Tables S1 and S2 summarize the differences in the crystal structures and model structures of CarH, GLM, and EAL. CarH-DS is a tetramer, more specifically, it is a dimer-of-dimers type tetramer. In this study, the primary focus is the molecular activity in the
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors acknowledge the Cardinal Research Cluster (CRC) at the University of Louisville for providing access to high performance computing facilities.
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