Elsevier

Inorganica Chimica Acta

Volume 468, 1 November 2017, Pages 252-259
Inorganica Chimica Acta

Research paper
Electrochemical study of an electron shuttle diheme protein: The cytochrome c550 from T. thermophilus

https://doi.org/10.1016/j.ica.2017.05.009Get rights and content

Highlights

  • The redox properties of a diheme protein from Thermus thermophilus and its two domains are studied.

  • EPR measurements suggest the existence of cooperative interactions between the two hemes.

  • This very versatile protein interacts with its partners either through electrostatic or hydrophobic interactions.

Abstract

Cytochrome c550, a diheme protein from the thermophilic bacterium Thermus thermophilus, is involved in an alternative respiration pathway allowing the detoxification of sulfite ions. It transfers the two electrons released from the oxidation of sulfite in a sulfite:cytochrome c oxidoreductase (SOR) enzyme to heme/copper oxidases via the monoheme cytochrome c552. It consists of two conformationally independent and structurally different domains (the C- and N-terminal) connected by a flexible linker. Both domains harbor one heme moiety. We report here the redox properties of the full-length protein and the individual C- and N-terminal fragments. We show by UV/Vis and EPR potentiometric titrations that the two fragments exhibit very similar potentials, despite their different environments. In the full-length protein, however, the N-terminal heme is easier to reduce than the C-terminal one, due to cooperative interactions. This finding is consistent with the kinetic measurements which showed that the N-terminal domain only accepts electrons from the SOR. Cytochrome c552 is able to interact with its partners both through electrostatic and hydrophobic interactions as could be shown by measuring efficient electron transfer at gold electrodes modified with charged and hydrophobic groups, respectively. The coupling of electrochemistry with infrared spectroscopy allowed us to monitor the conformational changes induced by electron transfer to each heme separately and to both simultaneously.

Introduction

Biological energy conversion processes such as respiration and photosynthesis rely on efficient inter-and intra-protein electron transfer (ET) reactions [1], [2], [3]. In both the photosynthetic and respiratory chains, the transport of electrons between the enzymes embedded in the membrane is accomplished either by molecules freely diffusing in the membrane, the quinones, or water soluble electron carrying proteins, such as cytochrome (cyt) c. This small monoheme protein (12.5 kDa) [4], [5], [6], which transfers electrons between cyt bc1 complex and cyt c oxidase in the mitochondrial respiratory chain, has long served as a model system for mechanistic studies of protein ET processes. Important insight into the distance dependence and reorganization energy of the ET process has been gained from protein film voltammetry studies of cyt c immobilized on electrodes modified with various functional groups including alkyl [7], pyridyl [8], [9], amino [10], [11], carboxyl [12], [13], [14], l-cysteinyl [15] and hydroxyl [16]. In particular, studies at metallic surfaces modified with ω-carboxyl alkanethiols by a combined approach of electrochemistry, surface-enhanced and time-resolved spectroscopic techniques have provided a full image of the electron transfer dynamics of electrostatically immobilized cyt c and have highlighted the important role of electric field effects in the ET properties [17], [18], [19], [20], [21]. These surfaces have been designed to mimic the interaction between cyt c and cyt c oxidase, which is believed to involve a cluster of positively charged lysines residues on cytochrome c and several carboxylate groups of aspartic and glutamic acid residues on the oxidase [22], [23], [24].

Homologous electron carrier proteins from bacterial respiration chains have been less extensively studied. They often exhibit significantly different structural features, like for example the cyt c552 from the thermophilic bacterium Thermus thermophilus, which possesses only uncharged residues around the exposed heme edge [25], [26], [27]. Mainly hydrophobic interactions are thus believed to take place between cyt c552 and the corresponding cyt c oxidases in T. thermophilus [25], [28], [29]. Bacteria also use diheme proteins to transfer electrons between the respiratory enzyme complexes, such as the cyt c4, which are native electron donors of C family (cbb3) oxidases [30], [31]. They are the simplest model systems for multi heme proteins [31], [32], [33], [34]. Recently, a diheme electron carrier protein with 15% sequence identity only to cyt c4 was identified from T. thermophilus [35]. It was called cyt c550, on the basis of its spectral properties. It is involved in an alternative respiration pathway using sulfite ions instead of nicotinamide adenine dinucleotide as initial electron donor [35]. This pathway also allows the detoxification of the highly reactive sulfite ions. Cyt c550 receives electrons from the enzyme responsible for oxidation of sulfite, the sulfite:cytochrome c oxidoreductase (SOR), and further transfer them to the terminal cyt ba3 and caa3 oxidases through cyt c552. It is formed of two conformationally and structurally independent domains, each one harboring one heme c moiety [36]. These two domains will be referred as C-terminal (or cyt c550[C]) and N-terminal (or cyt c550[N]) in the following. Interestingly, the two domains exhibit different isoelectric points (5 and 7.97 for the cyt c550[C] and cyt c550[N] respectively), and thus opposite net charges at pH 7. It was established by stopped-flow UV/Vis experiments that the N-terminal domain only receives electrons from SOR, while both N and C-terminal are able to give them to cyt c552 [36]. This protein was thus proposed to function as an electron shuttle rather than an electron wire. Electrostatic forces seem to play an important role in the interaction between the N-terminal domain and both SOR and cyt c552, whereas hydrophobic residues are suggested to be involved in the interaction between the C-terminal domain and cyt c552.

Further insight into the function of cyt c550 can be obtained through the study of its redox properties. Hereafter we compare the voltammetric behavior at nanostructured gold electrodes of the full-length cyt c550 (23 kDa), as well as cyt c550[C] (14 kDa) and cyt c550[N] (9 kDa), which have been separately cloned and expressed [36], and discuss it in light of related systems, such as the cyt c4 and cyt c552. To take into account the distinct surface properties of these proteins, different modifications of the gold surface were probed. The redox potentials of the hemes were also determined by potentiometric titrations followed both by UV/Vis and EPR spectroscopies, in order to identify cooperative interactions between them. Finally, coupling of electrochemistry and infrared spectroscopic techniques allowed us to monitor the conformational changes occurring in the protein and the heme cofactor during redox reaction.

Section snippets

Chemicals

Sodium citrate, hydrogen tetrachloroaurate trihydrate, mercaptopropionic acid, cysteamine, 6-mercaptohexan-1-ol, 1-hexanethiol, 6-mercaptohexanoic acid, potassium phosphate dibasic trihydrate were purchased from Sigma and were used without further purifications.

Protein samples preparation

The cyt c550 and its individual domains were purified based on the methods detailed previously [35], [36] with the following modifications. After protein expression due to the T7 leaky expression and periplasmic protein extraction, the

Potentiometric titrations followed by UV/Vis spectroscopy

Fig. 1 shows the oxidized minus reduced UV/VIS difference spectra of full length cyt c550 (A), cyt c550[C] (B) and cyt c550[N] (C) obtained during a titration from −0.2 to +0.5 V. In the fully reduced form at −0.2 V, cyt c550 and cyt c550[C] exhibit a Soret band at 419 nm, a beta band at 520 nm and a composite alpha (α) band at 548 and 554 nm. Interestingly, for cyt c550[N], the Soret band is downshifted to 416 nm, and the α-band merges to one large peak at 550 nm. In the oxidized form at +0.5 V, the

Conclusion

Both voltammetric studies and potentiometric titrations show that the hemes of the cyt c550[C] and cyt c550[N] fragments have very close mid potentials values. In the full-length protein, however, EPR studies suggest that the N-terminal heme is easier to reduce than the C-terminal due to cooperative interactions. Interestingly, efficient electron transfer rates were observed on gold surfaces modified either with negatively-charged ω-carboxyl alkanethiols or a hydrophobic mixture of

Acknowledgements

FM and PH are grateful to the icFRC Labex, the CNRS and the University of Strasbourg for financial support. SA is a PhD student supported by a grant from the Libyan Ministry of Education. Part of this study was also facilitated by a HEA grant under the Programme for Research in Third-Level Institutions (PRTLI 5) of the University of Limerick. BSC is grateful to the EPR facilities available at the Aix-Marseille University EPR center, and to financial support from the French EPR network (RENARD,

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