Elsevier

Bioorganic Chemistry

Volume 109, April 2021, 104721
Bioorganic Chemistry

A new twist of rubredoxin function in M. tuberculosis

https://doi.org/10.1016/j.bioorg.2021.104721Get rights and content

Highlights

  • [1Fe-0S] metalloprotein is capable to support reactions catalyzed by cytochrome P450s.

  • Crystal structure of RubB from M. tuberculosis has a typical rubredoxin fold.

  • RubB shuttles electrons from mycobacterial reductases FdR and FprA to at least three CYPs.

  • RubB belongs to rubredoxins with low redox potential (-264 mV ± 17 vs. Ag/AgCl)

  • Interaction of RubB with redox partners occurs via multipoint or/and transient complexes.

Abstract

Electron transfer mediated by metalloproteins drives many biological processes. Rubredoxins are a ubiquitous [1Fe-0S] class of electron carriers that play an important role in bacterial adaptation to changing environmental conditions. In Mycobacterium tuberculosis, oxidative and acidic stresses as well as iron starvation induce rubredoxins expression. However, their functions during M. tuberculosis infection are unknown. In the present work, we show that rubredoxin B (RubB) is able to efficiently shuttle electrons from cognate reductases, FprA and FdR to support catalytic activity of cytochrome P450s, CYP124, CYP125, and CYP142, which are important for bacterial viability and pathogenicity. We solved the crystal structure of RubB and characterized the interaction between RubB and CYPs using site-directed mutagenesis. Mutations that not only neutralize single charge but also change the specific residues on the surface of RubB did not dramatically decrease activity of studied CYPs. Together with isothermal calorimetry (ITC) experiments, the obtained results suggest that interactions are transient and not highly specific. The redox potential of RubB is −264 mV vs. Ag/AgCl and the measured extinction coefficients are 9931 M−1cm−1 and 8371 M−1cm−1 at 380 nm and 490 nm, respectively. Characteristic parameters of RubB along with the discovered function might be useful for biotechnological applications. Our findings suggest that a switch from ferredoxins to rubredoxins might be crucial for M. tuberculosis to support CYPs activity during the infection.

Introduction

Tuberculosis was the first infectious disease to be declared a global health emergency by the World Health Organization; tuberculosis causes more than 1.4 million deaths every year [1]. Multiple mechanisms allow Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis, to survive within macrophages. These mechanisms include production of the antioxidant molecule mycothiol [2], activation of the catalase-peroxidase katG and superoxide dismutases sodA and sodC [3], and synthesis of truncated hemoglobins [4]. In phagosomes, Mtb resides in an acidic environment, thus requiring mechanisms to sustain growth under oxidative and acidic stresses. Moreover, during granuloma formation Mtb experiences drastic iron deprivation that influences the intensity of iron-sulfur clusters assembly in redox proteins [5]. The presence of a variety of genes that encode [3Fe-4S] and [4Fe-4S] ferredoxins and [1Fe-0S] rubredoxins identified in Mtb suggests the important role of iron-containing proteins in maintaining redox homeostasis. We hypothesized that rubredoxins, which have been suggested to be part of an evolutionary chain between ferredoxins and flavodoxins [6], might be important in reactions catalyzed by cytochrome P450 (CYP) proteins.

Rubredoxins are small (~6 kDa) iron–sulfur proteins that are crucial for oxidative stress responses. They rapidly transfer metabolic reducing equivalents to oxygen or reactive oxygen species and act as electron carriers in many biochemical pathways [7]. The Mtb genome contains a highly conserved operon comprised of two tandemly arranged rubredoxin-encoding genes, RubA (Rv3251c) and RubB (Rv3250c), and the gene alkB (Rv3252c), which encodes an alkane hydroxylase. The gene products are probably involved in alkane and fatty acid metabolism. Our bioinformatic analysis of rubredoxins across different phylogenetic groups (Fig. S1, S2) showed that RubA is conserved in Actinobacteria and is specific for this family, whereas RubB is observed in gamma- and beta-proteobacteria. Large and distinctive rubredoxin families are found in Bacteroidetes and Firmicutes phyla; these genes group with genes encoding primitive archaeal rubredoxins, and plant-specific rubredoxins, demonstrating evolutionary conservation of rubredoxin scaffold.

Expression of both Mtb rubredoxins is induced under iron starvation [5] and during iron chelator 2,2′-bipyridyl application [8]. Oxidative stress induced by the alkaloid ascidemin or nitrosative stress caused by diethylenetriamine/NO or a mixture of S-nitrosoglutathione and potassium cyanide strongly induce expression of rubredoxins in Mtb culture. Finally, rubredoxins are also induced by culture of Mtb in acidic pH [9]. Notably, rubredoxin induction is often accompanied by inhibition of ferredoxin expression [10].

Induction of rubredoxin expression during oxidative and acidic stresses as well as under iron starvation might be due to the physical–chemical properties of rubredoxins. Rubredoxins need only one iron ion, which is beneficial during residence within macrophages in which microelements are deficient. Further, rubredoxins are usually of lower molecular mass than ferredoxins and do not require scaffold proteins, thus requiring fewer biosynthetic resources than ferredoxins. Moreover, rubredoxin activity is not inhibited by oxidative, acidic and temperature stresses, which may be crucial in the face of a host immune response [10], [11]. This evidence suggests that rubredoxins are more advantageous redox partners for CYPs than ferredoxins.

To test this idea, we purified RubB protein and measured enzymatic activity of three Mtb CYPs in the reconstituted system, containing RubB and different reductases as redox-partners. Our results demonstrate that RubB supports CYP-dependent catalysis. We also performed biophysical characterization of RubB including crystal structure determination. Site-directed mutagenesis was used for mapping of the protein–protein interactions within the RubB – CYP complex. Based on data obtained using purified RubB mutants, we suggest that the RubB interaction with CYPs is transient and not highly specific, as point mutations on the surface of RubB does not dramatically affect CYP activity. Overall, our results provide new insights into electron transfer within different classes of redox proteins in Mtb and suggest that these interactions might be important switch mechanisms during different stages of Mtb infection.

Section snippets

Expression, purification and spectral properties of RubB

RubB was cloned into the pET11a vector and overexpressed in Escherichia coli. According to a previous report, both iron and zinc-substituted rubredoxins are produced during heterologous expression in E.coli [12]. As binding of zinc and iron to RubB might be a competitive process [13], we provided extra iron during culture to obtain primarily the iron-containing form. RubB was purified to a homogenous state using sequential ion-exchange and size-exclusion chromatography. Based on size-exclusion

Discussion

Rubredoxins perform electron transfer as separate proteins. However, rubredoxin domains are found to have diverse roles within proteins from different families. Their functions vary from the adaptation to a changing redox environment [41] to the maintaining of the overall protein stability [42] and are associated with different developmental processes [43]. In present study, we demonstrated that the Mtb rubredoxin RubB is a potent redox partner for Mtb cytochrome P450s. We reconstituted the

Cloning and expression of Mtb RubB

The RubB (Rv3250c) gene was amplified from Mtb genomic DNA. The PCR product was ligated into the expression vector pET11a.

E. coli C43 competent cells were transformed with pET11a containing RubB. Transformed cells were screened on Petri dishes with LB-agar containing ampicillin (100 µg/ml). An overnight culture (3 ml) was used to inoculate 0.5 L of TB-medium containing 100 mM potassium-phosphate buffer, pH 7.4, and 100 µg/ml ampicillin. The mixture was incubated in a thermostated orbital shaker

CRediT authorship contribution statement

Tatsiana Sushko: Investigation, Writing - original draft. Anton Kavaleuski: Investigation. Irina Grabovec: Investigation. Anna Kavaleuskaya: Investigation. Daniil Vakhrameev: Investigation, Writing - original draft. Sergey Bukhdruker: Investigation, Writing - review & editing. Egor Marin: Investigation. Alexey Kuzikov: Investigation. Rami Masamrekh: Investigation. Victoria Shumyantseva: Methodology, Investigation. Kouhei Tsumoto: Methodology. Valentin Borshchevskiy: Methodology, Writing -

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.

Acknowledgments

This study was inspired by personal communication with Prof. Trevor Forsyth (The Institut Laue–Langevin, France). This work was supported by a joint grant received from Belarusian Republican Foundation for Fundamental Research, B20R-061 and Russian Foundation for Basic Research, 20-54-00005. V.B. is supported by the Ministry of Science and Higher Education of the Russian Federation (agreement #075-00337-20-03, project FSMG-2020-0003). Electrochemical experiments were performed in the framework

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      In total 5 different RubB mutants were cloned, expressed, purified and analyzed. As described previously, D44A, Q11V, S41V and E24A mutations did not induce significant structural changes in RubB [8]. A CD spectrum of D25ins mutant is typical for rubredoxins with a double minimum at 204 nm and 227 nm indicating that 14 AA insertion between Cys-coordinating iron did not disturb the Fe-containing cluster (Fig. 2A).

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