Keywords: cytochrome, electron transfer, crystallization, diffraction

Introduction

The cytochromes are ubiquitous proteins present in all living organisms and involved in a variety of intracellular processes that are essential for life. Most notable is their participation in electron transfer reactions, usually as components of a complex reaction pathway, necessary for the production of energy either through oxidation of metabolites or via photosynthesis. Cytochromes are members of a larger class of proteins, known as hemoproteins. The hemoproteins derive their name from the presence of one or more iron porphyrin prosthetic groups (called as hemes). Besides cellular bioenergetics, the heme is also involved in ligand binding reactions necessary for oxygen transport [1].

Compared with other biologically active molecules, cytochromes are some of the simplest bioinorganic compounds considering of molecular weight and structure. The active center of cytochromes is the heme group [2]. It consists of a porphyrin ring chelated to an iron atom. The porphyrin ring is a macrocyclic pyrrole system with conjugated double bonds. These compounds undergo chemical oxidation and reduction, cycling between ferrous (Fe²⁺) and ferric (Fe³⁺) forms, in contrast to the hemoglobin, where the iron is normally in the ferrous (Fe²⁺) state.

Our cytochrome is characterised by alpha-peak wavelength of 553 and a molecular mass of 25 kDa. The absorption spectrum (performed by UV-visible absorption spectroscopy [3]) presents distinct split alfa band, and a very low alfa to beta ratio. The protein consist of two heme molecules in single polypeptide chain with classical Cys–X–Y–Cys–His heme binding sites. The fifth heme iron ligand is always provided by a histidine residue [2]. Cytochrome is located probably in bacterial periplasmic space of Thiocapsa roseopersicina cell wall. The established properties of this hemoprotein indicate that it belongs to the c₄ family [3] of diheme cytochromes. It is the first cytochrome of this class that comes from an anaerobic organism. Due to its important function, it is of essential interest to study structural features of cytochromes using X-ray crystallography.

Materials and crystallization methods

Cytochrome c₄ (cyt c₄) from the purple photosynthetic bacterium Thiocapsa roseopersicina was isolated and purified according to [3]. This bacterium has four different hydrogenases and three different cytochromes. The cyt c₄ has been studying by crystallographic, proteolytic [4] and spectroscopic methods. Cyt c₄ was crystallized using standard crystallization methods based on vapor diffusion (hanging and sitting drops [5]) and advanced crystallization method based on the counter-diffusion (crystallization in capillaries [6]).

Figure 1: A, B – Pseudocrystals of cyt c₄ and C – crystal of cyt c₄ constructed together with AS.

Initial crystallization trials with ammonium sulfate (AS) yielded pseudocrystals as red thin plate [Figure 1] with components from 0.1 M sodium chloride and 0.1 M citric acid pH 6.0 in the reservoir solution. Ranging pH value higher than 7.5 the phase separation of protein appeared.

Crystallization trials were performed at 20 °C. After fine-tuning crystallization conditions, the most suitable concentration of protein (10–15 mg/ml) and the percentage of precipitation agent were found. The first suitable crystal growth was observed at pH 6.0 [Figure 2] using the addition of metal ions – Cu²⁺, Cd²⁺, Co²⁺, Ba²⁺ (from Hampton Research Additive Screen HR2–428). Cyt c₄crystals were grown in capillaries when the precipitating system contacts the protein solution because a wave of supersaturation was triggered.

Figure 2: A, B – Crystals of cyt c₄

Diffraction measurement

The monocrystals of cyt c₄ were tested at the home source diffractometer at LEC (University of Granada) and measured at synchrotron DESY (Hamburg) in the loops and in the capillaries directly. Structure of cyt c₄ will be solved using molecular replacement method.

Colored crossbred plates of holoprotein crystals with dimensions of approximately 230 x 40 x 20 μm grew within 3–4 days under several conditions. Protein crystals grown in capillaries were measured directly at synchrotron DESY (Hamburg), beamline X11. Crystallization conditions are now optimized in order to prepare monocrystals of cyt c₄ suitable for X-ray structural analysis.

References

1. G. Moore, F. Pettigrew: Cytochromes c, Springer-Verlag, Berlin (1987)

2. T. Yamanaka: The Biochemistry Of Bacterial Cytochromes, Japan Scientific Societies Press, Tokyo (1992)

3. Cs. Bagyinka, R. M. M. Branca: unpublished data (2005)

4. J. Carey: Methods Enzymol., Vol. 328, 449 (2000)

5. T. M. Bergfors: Protein Crystallization. Techniques, Strategies and Tips. International University Line, La Jolla, USA (1999)

6. F. J. López-Jaramillo, J. M. García-Ruiz, J. A. Gavira, F. Otálora: J. Appl. Cryst. 34, 365-370 (2001)

Acknowledgements

This work is supported by grants of the Ministry of Education of the Czech Republic (by grants KONTAKT ME640, MSM6007665808 and AVOZ60870520) to I.K.S. and by the EMBL-Hamburg Strategy Fund. We are grateful to X11 Consortium for Protein Crystallography for access to their facility. We would like to thank Dr. Matthew Groves and Dr. Victor Lamzin (EMBL Hamburg) for help with the access to EMBL and processing at X11 beamline and Dr. Jose A. Gavira for his help with crystals testing at the LEC diffractometer. This research was also supported by the Ministry of Education, Youth and Sports of
the Czech Republic (MSM6007665808, LC06010) and by the Academy of Sciences of
the Czech Republic (Institutional research concept AVOZ60870520).