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Hyscore spectroscopy of the cytochrome b 559 of photosystem ii reaction centre

Cytochromes are electron transfer heme proteins that are involved in many biochemical processes and exhibit a large variety of redox potential. These characteristics allow them to act as electron carriers in different metabolic reactions and other processes in living organisms. The relationship between the electronic structure of the heme group (the final acceptor of the first donor in the redox reaction of cytochromes) and the reaction mechanism is not well understood in most cases.

As the ferric state of the cytochromes is paramagnetic, Electron Paramagnetic Reso-nance (EPR) techniques have been used to characterize this kind of proteins [1]. Continuous wave (cw-EPR) spectroscopy gives useful information about the unpaired electronic distribution in the heme centers [1]. Nevertheless, no information about the hyperfine and quadrupolar interactions with the neighboring nuclei can be obtained with this conventional technique because of its lack of resolution. With the aim of overcome this limitation ENDOR and, more scarcely, electron spin echo modulation (ESEEM) techniques have been applied to study different heminic systems, both in hemeproteins and heme model systems [2-3].

It has to be taken into account that in hemoproteins the Fe 3+ paramagnetic entity is surrounded by many magnetic nuclei (five or six nitrogen and several protons) interacting with the unpaired electron. Selective isotopic substitution would lead to a direct assignment of the signal to particular nuclei but in the practice this is not always achievable when working with biological materials. In this case the use of model systems can be helpful. Moreover, protein samples are orientationally disordered systems in most cases. Thus, the corresponding ENDOR as well as one-dimensional (1D) ESEEM spectra strongly overlap and thus are difficult to disentangle.

On the other hand, the two-dimensional (2D) ESEEM HYSCORE spectroscopy introduced some years ago has proved to be useful to study weak hyperfine interaction with many nuclei in orientationally disordered systems [4]. Nonetheless, this potential has not yet been exploited to study heme systems.

We have examine the ferric state of cytochrome (Cyt) b 559 from spinach by means of the HYSCORE technique. Cyt b 559 is an hemeprotein constituent of the photosystem II (PSII) reaction center (RC), which is bound to the D1 and D2 polypeptides of the PSII RC. We have studied Cyt b 559 in D1-D2-Cyt b 559 reaction center complexes, which are the minimum PSII complexes that are able to perform efficient light induced primary charge separation. This hemeprotein consists of two small polypeptides, the a and b subunits. Two histidine residues placed axially respect to the phorphyrin ring with the hydrophobic domain of each polypeptide act as ligands of the heme iron.

Cyt b 559 was characterized by cw-EPR spectroscopy a few years ago [5]. Its spectra are typical of a low spin heme iron and can be described with an fictitious spin S = ½ and an orthorrombic g -tensor with the following principal values: g x » 1.5, g y » 2.3, g z » 3.0. Slight modifications of the g z -value depending on the preparation and purification have been described. More recent studies relate these minor modifications in the cw-EPR spectra with changes between different redox forms of Cyt b 559 [5]. More recent studies performed in our laboratory point out that the conditions of the detergent used to stabilize the PSII RC sample is the driving force that induces the observed changes in the cw-EPR spectra [6].

With those antecedents a HYSCORE study of Cyt b 559 was undertaken. In this case, it is not possible to work with selectively labeled Cyt b 559 . Thus we have also studied heme model compounds that have similar cw-EPR and HYSCORE spectra. In these model compounds two imidazol axial ligands mimic the histidine residues. By selective isotopic substitution of iron bond nitrogen atoms in these simpler systems a direct assignment of the HYSCORE signals was done. Because of the similarity of the spectra this assignment can be extended to the Cyt b 559 .

The high anisotropy of the cw-EPR spectra allows making orientation selection HYSCORE spectra. Different spectra for magnetic field values corresponding to the principal values of the g -tensor were measured.

Figura 1: HYSCORE spectrum of a 14N natural abundance model compound measured at 10 K with an static magnetic field of 234 mT (gz-spectrum) the separation between the two fisrt pulse was t = 96 ns. The dq-dq correlation due to N-Im and N-Hem are indicated.

As an example, the HYSCORE spectrum measured with a magnetic field corresponding to g z (234 mT) in the model compound with a natural content of 14 N is given is figure 1. By comparison with the HYSCORE spectra of 15 N selectively labeled in the porphyrin ring, in the imidazol moiety or in both, the assignation of the observed signal to each type of nitrogen atoms was done

The HYSCORE spectra allow obtaining the corresponding nuclear splitting of both types of nitrogen atoms and then, the principal values of the hyperfine and quadrupolar coupling tensor and the orientation of their principal axes respect to the g -tensor.

As all the heme nitrogen atoms are equivalent in the g z spectra it is followed that the z -principal axis of the g -tensor is normal to the heme plane. Consequently, the orientation of the g -tensor principal axes respect to the molecule is determine by the angle, Q , that the X-axis makes with a Fe-N-Hem bond direction. From the quadrupolar tensor of the N-Hem atoms is estimated that 20 ° < Q < 38 ° . This is illustrated in figure 2.a where the orientation of the g -tensor X -principal axis in the porphyrine plane is indicated

Figura 2: (a) Orientation of the g-tensor X-principal axis in the porphyrine plane; dashed lines indicate a range for possible orientation and the continuous arrowed line shows the mean orientation. (b) The thick line indicates the orientation of the imidazol plane; the range of possible oreintations is in between the dashed lines.

From the analysis of the quadrupolar tensor for N-Im atoms the orientation of the imidazol plane is obtained. It is depicted in figure 2.b. Furthermore the g -tensor principal values strongly suggest that both imidazol plane are parallel.

An analysis of the g -tensor using the Taylor´s model [1] indicates that the unpaired electron density is mainly in a d y´z like-orbital which is normal to the heme ring and to the imidazol plane. It is also illustrated in figure 2.b.

As far as the hyperfine interaction with the nitrogen atoms is concerned the obtained results indicate that this interaction is mainly isotropic and the unpaired electronic orbital have a strong non-bonding character.

The strong similarity between the spectra of the model compound and Cyt b 559 lead to extent these conclusions to the cytochrome. In particular the structure of the heminic center in this protein should be the same as the one depicted in figure 2. It is worth of noting that in spite of the observed principal g -values modification no differences are found in the interaction parameters when detergent conditions used to stabilize Cyt b 559 samples change.

As it has been commented above, the hyperfine data indicate that the unpaired electron is localized in confined iron orbital with a negligible mixture of p-orbitals. This makes very unlikely that any substrate or reactive can be located close enough to the orbital occupied by the exchangeable electron for a direct one-step electron exchange. The transfer mechanism could be better understood if it were a multistage complex process where the metal in the heme group acts as a final electron reservoir. So, the g -tensor principal values would mainly depend on the metal electronic structure and near environment whereas measured redox potential would rather be related with the accessibility of mediators and the actual reaction process in the experiment. Such processes will depend in a complex way on the whole protein conformation. That would explain that there is not a direct relationship between the cw-EPR signal and redox potential as it was assumed before [5].

Finally we want to point out that this assertion could be considered as a more general conclusion being extended to other cytochromes. Anyway, this work proves the strong potentiality of the HYSCORE spectroscopy to obtain relevant information about the structure and function of metalloproteins.

Principal publication
I. García-Rubio, J.I. Martínez, R. Picorel, I. Yruela, P.J. Alonso, J. Am. Chem. Soc. 2003, 125 , 15846.

Acknowledgements
This work was supported by the CONSI+D (DGA, local government of Aragón) under contract P111/2001. 

References
[1] C.P.S. Taylor, Biochem. Biophys. Acta 1977, 491 , 137.
[2] N.D. Chasteen and P.A. Snetsinger. In Physical Methods in Bioinorganic Chemistry. Spectroscopy and Magnetism . L. Que Editor University Science Books. Sausalito (CA, USA ) 2000. ch 4.
[3] F.A. Walker. Inorg. Chem . 2003, 42 , 4526.
[4] J.J. Shane, P. Höfer, E.J. Reijerse, E. de Boer, J. Magn. Reson . 1992, 99 , 596.
[5] D.M. Stewart, G.w. Brudvig. Biochem. Biophys. Acta 1998, 1367 , 63.
[6] I. Yruela, I. García-Rubio, M. Roncel, J.I. Martínez, V.M. Ramiro, J.M. Ortega, P.J. Alonso, R. Picorel, Photochem. Photobiol. Sci . 2003, 2 , 437.

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©2017 Instituto de Ciencia de Materiales de Aragón | Tfno: 976 761 231 - Fax: 976 762 453
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