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Efficient and selective isotopic labeling of hemes to facilitate the study of multiheme proteins
Bruno M. Fonseca1, Ming Tien2, Mario Rivera3, Liang Shi4, and Ricardo O. Louro1
1Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
2Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, USA
3Department of Chemistry, University of Kansas, Lawrence, KS, USA
4Microbiology Group, Pacific Northwest National Laboratory, Richland, WA, USA
BioTechniques, Vol. , No. , April 2012, pp. 1–7
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Method Summary

Here we bring together tools in molecular biology, cloning, organic synthesis, and metabolic regulation to assemble a coherent strategy for the efficient isotopic labeling of specific carbon atoms in the hemes of what are unarguably the most challenging heme proteins, the multiheme cytochromes c. This method provides a rationally designed window into the functional and structural properties of these proteins, which in recent times have become the focus of great attention due to their role in biogeochemical processes and potential for bioremediation of contaminated sites and bioenergy production from renewable sources.


Specific isotopic labeling of hemes provides a unique opportunity to characterize the structure and function of heme-proteins. Unfortunately, current methods do not allow efficient labeling in high yields of multiheme cytochromes c, which are of great biotechnological interest. Here, a method for production of recombinant multiheme cytochromes c in Escherichia coli with isotopically labeled hemes is reported. A small tetraheme cytochrome of 12 kDa from Shewanella oneidensis MR-1 was used to demonstrate the method, achieving a production of 4 mg pure protein per liter. This method achieves, in a single step, efficient expression and incorporation of hemes isotopically labeled in specific atom positions adequate for spectroscopic characterization of these complex heme proteins. It is, furthermore, of general application to heme proteins, opening new possibilities for the characterization of this important class of proteins.

Heme proteins hold a special place in the development of modern biochemistry. Hemoglobin is aptly considered an honorary enzyme despite its physiological role as a diatomic gas transporter (1). Indeed, heme proteins perform ubiquitous cell functions such as electron transfer (cytochromes) (2-4), energy transduction (cytochrome c oxidase) (5, 6), catalysis (P450 and peroxidases) (7-10), or molecular sensing (chemotactic proteins) (11). One class of heme proteins has recently gathered considerable attention, being the focus of a Biogeochemistry Grand Challenge of the U.S. Department of Energy. These are multiheme cytochromes c, which mediate electron transfer at the microbe-mineral interface in geological settings and at the microbe-electrode interface in bioelectrochemical devices (12, 13). These cytochromes have been shown to play major roles in cellular respiration and to exist in almost all major groups of Bacteria and Archaea (14). In some of these organisms, such as representatives of Geobacter, Shewanella, Anaeromyxobacter, or Desulfovibrio genera, the number of multiheme cytochromes is so elevated that it corresponds to a high percentage of their proteome, having elicited the creation of the term “cytochromome” (15, 16). For Geobacter and Shewanella, many of these are known to be essential for the extracellular respiration that is at the core of electricity production in microbial fuel cells (17).

Given their biological importance, considerable effort has gone into the development of efficient methods for their recombinant production to facilitate their molecular characterization and/or their use in medical or biotechnological applications (12, 16-18). In comparison to other types of cytochromes, multiheme cytochromes c are more difficult to express correctly on two accounts: the various hemes must be covalently attached, through thioether linkages, to the polypeptide chain at the CXX(XX)CH binding site; and also the correct distal axial ligand must be connected to the iron in the nascent protein. This is essential in order to obtain the native fold of the protein. For this to occur properly in Gram negative bacteria, specific molecular assembly helper proteins, collectively known as the cytochrome c maturation proteins CcmA-H (ccm cluster), are needed (19, 20). These proteins are responsible for the correct ligation of the heme to the apoprotein, while it is translocated to the periplasmic space. This cytochrome cbiogenesis system is denominated as system I and is the most complex of the presently known cytochrome c biogenesis systems, allowing the maturation of a variety of c-type cytochromes under different conditions (20-22).

Among many systems available for recombinant protein expression, the bacterium Escherichia coli is one of the most attractive hosts, due to the advantage of fast growth at a high density in an inexpensive medium, well-characterized genetics, and the availability of a large number of cloning vectors. With the insertion of a plasmid containing the ccm cluster (23, 24), E. coli becomes capable of expressing c-type cytochromes with correctly inserted hemes under aerobic conditions. Also, under these conditions, E.coli has the advantage of expressing only the recombinant c-type cytochromes. This simplifies considerably the protein purification procedure in comparison to other expression hosts described in the literature (25-29), which also produce their own native c-type cytochromes under the expression conditions used.

Multiheme cytochromes c are also more difficult to analyze with respect to their detailed functional properties, due to the multiple combinations of electron distribution that can occur among the various hemes. Since the heme cofactors are the functional components of multiheme proteins, their specific isotopic labeling is an attractive strategy to analyze the structure and function of these proteins. Toward this end, several approaches have been developed involving the supplementation of the growth media with specifically labeled heme cofactors (30, 31) or with isotopically labeled heme precursor δ-aminolevulinic acid (dALA) (31-33). Also, to guarantee that the uptake of these substituents is efficient, bacterial strains that are incapable of synthetizing hemes cofactors were created by deleting genes that are responsible for biosynthesis of dALA in the cell, such as the hemA gene (33, 34).

However, the methods presently published do not allow isotopic labeling of the hemes in multiheme c-type cytochromes (30-33), preventing the study of these more complex cytochromes. Here, a method that allows efficient expression of recombinant multiheme cytochromes c with specific isotopic labeling in the various hemes is reported. This method will bring an enormous advantage for the characterization of this important class of proteins by several spectroscopic techniques.

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