A bacterial 2[4Fesingle bond4S] ferredoxin as redox partner of the plastidic-type ferredoxin-NADP+ reductase from Leptospira interrogans

https://doi.org/10.1016/j.bbagen.2019.01.004Get rights and content

Highlights

  • We expressed, purified and characterized a 2[4Fesingle bond4S] ferredoxin from Leptospira interrogans.

  • The 2[4Fesingle bond4S] ferredoxin exchanges electrons with a plastidic ferredoxin-NADP+ reductase.

  • Ferredoxin recognition by ferredoxin-NADP+ reductase involves a unique structural subdomain.

  • We found a new redox partner of the plastidic-type ferredoxin-NADP+ reductases.

Abstract

Background

Ferredoxins are small iron-sulfur proteins that participate as electron donors in various metabolic pathways. They are recognized substrates of ferredoxin-NADP+ reductases (FNR) in redox metabolisms in mitochondria, plastids, and bacteria. We previously found a plastidic-type FNR in Leptospira interrogans (LepFNR), a parasitic bacterium of animals and humans. Nevertheless, we did not identify plant-type ferredoxins or flavodoxins, the common partners of this kind of FNR.

Methods

Sequence alignment, phylogenetical analyses and structural modeling were performed for the identification of a 2[4Fesingle bond4S] ferredoxin (LepFd2) as a putative redox partner of LepFNR in L. interrogans. The gene encoding LepFd2 was cloned and the protein overexpressed and purified. The functional properties of LepFd2 and LepFNR-LepFd2 complex were analyzed by kinetic and mutagenesis studies.

Results

We succeeded in expressing and purifying LepFd2 with its Fesingle bondS cluster properly bound. We found that LepFd2 exchanges electrons with LepFNR. Moreover, a unique structural subdomain of LepFNR (loop P75-Y91), was shown to be involved in the recognition and binding of LepFd2. This structural subdomain is not found in other FNR homologs.

Conclusions

We report for the first time a redox pair in L. interrogans in which a plastidic FNR exchanges electron with a bacterial 2[4Fesingle bond4S] ferredoxin. We characterized this reaction and proposed a model for the productive LepFNR-LepFd2 complex.

General significance

Our findings suggest that the interaction of LepFNR with the iron-sulfur protein would be different from the one previously described for the homolog enzymes. This knowledge would be useful for the design of specific LepFNR inhibitors.

Introduction

Ferredoxins (Fds) are a group of ubiquitous proteins containing iron-sulfur clusters. These proteins have been implicated in at least 18 metabolic pathways and as redox partners for enzymes that catalyze >75 different reactions [1]. Fds participate in electron transfers and contain different iron-sulfur clusters. Those of [2Fe-2S] structure are found in chloroplast Fds operating as electron donors to enzymes such as nitrite reductase and glutamate synthase [2]. They are also found in many animal tissues, acting as electron donors to cytochrome P450 [3]. [3Fesingle bond4S] and [4Fesingle bond4S] cluster Fds were found in bacteria. Bacterial Fds may also contain two [4Fe-4S] or [3Fesingle bond4S] clusters or [4Fesingle bond4S] [3Fesingle bond4S] cluster combination, and operate mainly as low potential electron transfer proteins [4].

Fd from Clostridium pasteurianum, which is involved in nitrogen fixation, was the first isolated in 1962. [5]. Subsequently, a Fd from spinach chloroplasts was implicated in photoreduction of NADP+ [6]. Afterward, many novel Fds were found, and studies were focused in the structure elucidation of these proteins and their active sites. The obtained results have shown that the structural and functional diversity of Fds is far greater than expected by the small number of different cluster types. This finding suggested an essential role of the peptide moiety in determining the properties of the cluster.

The Fds with [2Fe-2S] clusters can be grouped into Fds involved in photosynthesis and those that participate in other different metabolic processes [4]. The Fds of the first group function as terminal electron acceptor from photosystem I in green plants, algal, and cyanobacterial photosynthesis. The [2Fesingle bond2S] center receives one electron and then the Fd reduces the flavin moiety of ferredoxin-NADP+ reductase (FNR). This reaction proceeds through two one-electron transfer steps with the subsequent generation of NADPH [7]. Chloroplast Fds are also implicated in several Fd-dependent reactions such as the reduction of nitrite to ammonia, nitrogen fixation, glutamate synthesis, sulfur assimilation, thioredoxin oxidoreduction and lipid desaturation [1]. The redox potentials of [2Fe-2S] Fds range between −305 and − 455 mV [8] and their pI are approximately 3 to 4.

Typically, redox potentials of approximately −400 mV are observed for [4Fesingle bond4S] Fds [9,10]. However, the actual potentials range from −280 mV, reported for Bacillus stearothermophilus Fd [11], to the unusually low value of −645 mV measured for the [4Fesingle bond4S] cluster of the [4Fesingle bond4S][3Fe-4S] Fd from Azotobacter vinelandii [12,13].

Generally, low-potential Fds function as electron carrier proteins and are found in all organisms which are consuming or producing hydrogen (for a review see [14]). Many organisms use these Fds for different purposes, as low potential electron carriers, particularly in anaerobic metabolism and they have probably more different functions than any other iron-sulfur protein [15].

The alignment of low-potential bacterial 2[4Fesingle bond4S] Fds shows high sequence similarities between the amino acid sequences, particularly in their N-terminal ~60 amino acids. They differ mainly in the C-terminal sequence following the coordination motif of the second cluster.

The Fds are commonly recognized substrates of the ferredoxin-NADP+ reductase. We found that FNR from Leptospira interrogans (LepFNR) belongs to the plastidic class of FNR at variance of all other bacterial enzymes [16,17]. We performed a detailed search of Fd homologs in the Leptospira genome, and we have not identified any plant-type Fd, the common partners of the plastidic class FNR. Moreover, experimental evidence showed that Fd from pea is not a good substrate for LepFNR [18]. Looking for the physiological substrate of this reductase we identified two putative Fd coding sequences, LA4086 (LepFd1) and LB107 (LepFd2), in the genome of L. interrogans. In previous studies, we found that LepFd1 corresponds to a [2Fesingle bond2S] Fd with thioredoxin-like fold and that it is not able to exchange electrons with LepFNR [19]. In this work, the gene encoding for LepFd2 was cloned and expressed in Escherichia coli cells, and the recombinant Fd was purified to homogeneity and characterized. By sequence and spectroscopy analyses we determined that it corresponds to a bacterial type 2[4Fesingle bond4S] Fd. The phylogenetic analysis indicates that Fds from the Leptospira genus clustered as an independent taxon and the closest sequences seem to be that from Planctomycetes. Also, Leptospira Fds are further subdivided into groups that may be associated with pathogenicity of the bacteria. To infer the metabolic role of LepFd2 we studied its functional properties. We found that the LepFd2 can receive electrons from the plastidic-type FNR found in the pathogen, suggesting that the iron-sulfur protein may be one of the physiological substrates of this reductase. Moreover, by mutagenesis studies, we determined that a unique structural subdomain of LepFNR, not found in other homologs, is involved in the recognition and binding of LepFd2.

Section snippets

Sequence alignment and phylogeny of LepFd2

A BLASTp search [20] was performed against the non-redundant protein sequences database available at NCBI, the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov, March 2017). The software Uclast was used to reduce redundancy within the protein set and to extract representative sequences [21]. Then, incomplete sequences and those that do not contain the typical amino acid clusters for [Fesingle bondS] binding were removed. Redundant sequences from Leptospira genus were added

Sequence alignment and phylogeny of LepFd2

A BLASTp search [20] was performed against the non-redundant protein sequences database available at NCBI, the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov, March 2017). A total of 14,096 sequences were recovered. The software Uclast was used to reduce redundancy within this protein set and to extract representative sequences [21]. Then, incomplete sequences, extremely short proteins and those that do not contain the typical amino acid for [4Fesingle bond4S] clusters

Conclusions

In this work, we studied and characterized a new substrate of the FNR found in the pathogenic bacterium L. interrogans, a 2[4Fesingle bond4S] Fd. It is the first report of this kind of Fd as redox partner of a plastidic-type reductase. Moreover, our findings suggest that interaction of LepFNR with the iron-sulfur protein would be different from the one previously described for the homolog enzymes and that a particular subdomain of LepFNR composed by the loop P75-Y91 is involved in the recognition and

Funding

This work was supported by grants from PICT-2015-2955, ANCYPT, Ministerio de Ciencia, Tecnología e Innovación Productiva, Argentina (www.agencia.mincyt.gov.ar); PIP 112–201201-00345-CO from CONICET, Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina (www.conicet.gov.ar) and, 1BIO346 and BIO497 from the University of Rosario, Argentina (www.unr.edu.ar.).

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