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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
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Moreover, to confirm that the ΔhemA E. coli LS543 strain is capable of expressing larger and more complex multiheme cytochromes c, an expression vector containing the gene of the decaheme cytochrome MtrA from S. oneidensis MR-1 of approximately 37 kDa, was used. Figure 1B shows that the resulting ΔhemA E. coli LS544 strain is also capable of expressing this protein, opening the door to the detailed characterization of these larger multiheme c-type cytochromes.

The auto-induction method developed by Studier (41) showed the best expression yields compared with the other protein expression methods tested (Figure 1A). This approach has the advantage of allowing the induction to occur gradually. This gradual process is essential for correct incorporation of the hemes, and allows the cultures to reach higher cell densities, thereby increasing the protein yield. A yield of approximately 4 mg pure STC(D2N) per liter of cell culture was obtained. To the best of our knowledge, to present date, this is the highest yield obtained for isotopically labeled multiheme cytochromes (40) and is comparable to other strategies used for overexpressing nonlabeled multiheme cytochromes c (25, 26, 46).

Thus, this expression method allows the efficient production of specifically isotopic labeled hemes and also their correct incorporation into a multiheme cytochrome c (Figures 2 and 3).

Using 1,2-13C-labeled dALA causes the incorporation of 13C at the methyl groups at the periphery of the heme macrocycle and also at the β- and carboxylate carbons of the propionate groups (Figure 2A). Figure 2 shows that adventitious unlabeled carbons in the methyl positions is below the detection limit of NMR experiments as can be confirmed by verifying the lack of residual peaks at the center of each doublet in spectra obtained without 13C decoupling (Figure 2A). In low-spin paramagnetic cytochromes, the heme methyls are reasonably sharp and typically located in a clean spectral region in the 13C dimension (Figure 3). This allows for a simple identification of these NMR signals that facilitates their assignment, since the remainder of the protein contains 13C only at natural abundance (╛1%).

This now opens the possibility to characterize in detail the structure and function of multiheme cytochromes containing a large number of hemes thanks to the greater spectral dispersion obtained in the 13C frequency versus the 1H frequency (Figure 3). The position of the heme methyl signals in low-spin paramagnetic hemes can be used to determine the orientation of the axial ligands and the placement of the magnetic axes system associated with the unpaired electron (47). When a multiheme cytochrome is titrated, the position of the methyl signals changes in ways that can be related with the oxidized fraction allowing for the determination of the relative reduction potentials of the hemes (48). The specific 13C labeling enabled by the method reported here is further suitable for characterizing proteins of large size or containing paramagnetic centers (49), because it allows the use of direct heteronuclear detection experiments such as 13C-13C NOESY, which may be more suitable than 1H based experiments in these cases.

Also, since dALA is a versatile labeling source for hemes, with different labeled carbons in dALA, different kinds of information can be obtained. For instance, considering NMR applications, using 5-13C-labeled dALA, the heme carbons attached to the meso protons can be labeled. Measurements of the residual dipolar coupling (RDC) of these signals provide information on the relative spatial orientation of the hemes (50).

A further general advantage of the method described here when applied to NMR spectroscopy is that the need for a highly concentrated sample, or even a pure sample, may be eliminated. Under aerobic conditions E. coli only expresses the cytochrome of interest, assuring the specific and efficient use of the labeled dALA in the biosynthesis of the hemes for this protein. Therefore, the 13C NMR spectrum is dominated by the signals of the labeled hemes. This advantage may facilitate the future characterization of multiheme c-type cytochromes that are difficult to express and purify, such as those associated to cell membranes, and may also allow the in cell characterization of cytochromes.

In conclusion, a strategy to efficiently produce multiheme cytochromes labeled at selected carbons in the hemes was developed. The simplicity of the method and its ability to produce isotopically labeled multiheme c-type cytochromes with a yield comparable to that obtained from the expression of unlabeled proteins, makes this approach potentially applicable to many different heme proteins. This is true even for those cytochromes that are not of the c-type and therefore dispense the need for covalent attachment of the heme to the polypeptide chain. The methodology will also enable the detailed structural and functional characterization of large multiheme cytochromes. A detailed characterization of these proteins, which mediate microbe-mineral or microbe-electrode contact, is essential to develop rationally designed bioelectrochemical devices and bioengineered systems for bioenergy production and bioremediation of environmental contaminants (13).


The plasmid pEC86 used in this work was a gift from Prof. L Thöny-Meyer. B.M.F. is the recipient of a PhD fellowship from Fundação para a Ciência e Tecnologia (FCT; SFRH/BD/41205/2007). L.S. was supported by the Subsurface Biogeochemical Research program/Office of Biological and Environmental Research, U.S. Department of Energy. Research in the author's laboratories was supported by grants PTDC/BIA-PRO 098158/2008, MIT-Pt BS-BB/1014/2008 from FCT awarded to R.O.L. and a grant from the National Science Foundation (MCB-0818488) awarded to M.R. This work was also supported by FCT through grant PEst-OE/EQB/LA0004/2011. The NMR data were collected at The Portuguese National NMR Network (REDE/1517/RMN/2005), supported by “Programa Operacional Ciência e Inovação (POCI) 2010” and FCT.

Competing interests

The authors declare no competing interests.

Address correspondence to Ricardo O. Louro, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República-EAN, 2780-157 Oeiras, Portugal. e-mail: [email protected]">[email protected]

1.) Brunori, M. 1999. Hemoglobin is an honorary enzyme. Trends Biochem. Sci. 24:158-161.

2.) Paquete, C.M., and R.O. Louro. 2010. Molecular details of multielectron transfer: the case of multiheme cytochromes from metal respiring organisms. Dalton Trans. 39:4259-4266.

3.) Rodgers, K.R., and G.S. Lukat-Rodgers. 2004.Electron transfer: cytochromes. In J. McCleverty, and T.J. Meyer (Eds.) Comprehensive Coordination Chemistry II. Pergamon Press, Oxford:17-60.

4.) Gray, H.B., and J.R. Winkler. 2010. Electron flow through metalloproteins. Biochim. Biophys. Acta 1797:1563-1572.

5.) Konstantinov, A.A. 2012. Cytochrome c oxidase: intermediates of the catalytic cycle and their energy-coupled interconversion. FEBS Lett. 586:630-639.

6.) Yoshikawa, S., K. Muramoto, and K. Shinzawa-Itoh. 2011. Proton-pumping mechanism of cytochrome c oxidase. Annu. Rev. Biophys. 40:205-223.

7.) Poulos, T.L. 2010. Thirty years of heme peroxidase structural biology. Arch. Biochem. Biophys. 500:3-12.

8.) Rittle, J., and M.T. Green. 2010. Cytochrome P450 compound I: capture, characterization, and C-H bond activation kinetics. Science 330:933-937.

9.) Poulos, T.L. 1995. Cytochrome P450. Curr. Opin. Struct. Biol. 5:767-774.

10.) Wade, R.C., D. Motiejunas, K. Schleinkofer Sudarko, P.J. Winn, A. Banerjee, A. Kariakin, and C. Jung. 2005. Multiple molecular recognition mechanisms. Cytochrome P450-a case study. Biochim. Biophys. Acta 1754:239-244.

11.) Pokkuluri, P.R., M. Pessanha, Y.Y. Londer, S.J. Wood, N.E. Duke, R. Wilton, T. Catarino, C.A. Salgueiro, and M. Schiffer. 2008. Structures and solution properties of two novel periplasmic sensor domains with c-type heme from chemotaxis proteins of Geobacter sulfurreducens: implications for signal transduction. J. Mol. Biol. 377:1498-1517.

12.) Lovley, D.R. 2006. Bug juice: harvesting electricity with microorganisms. Nat. Rev. Microbiol. 4:497-508.

13.) Summers, Z.M., H.E. Fogarty, C. Leang, A.E. Franks, N.S. Malvankar, and D.R. Lovley. 2010. Direct exchange of electrons within aggregates of an evolved syntrophic coculture of anaerobic bacteria. Science 330:1413-1415.

14.) Sharma, S., G. Cavallaro, and A. Rosato. 2010. A systematic investigation of multiheme c-type cytochromes in prokaryotes. J. Biol. Inorg. Chem. 15:559-571.

15.) Londer, Y.Y., S.E. Giuliani, T. Peppler, and F.R. Collart. 2008. Addressing Shewanella oneidensis “cytochromome”: the first step towards high-throughput expression of cytochromes c. Protein Expr. Purif. 62:128-137.

16.) Alves, A.S., C.M. Paquete, B.M. Fonseca, and R.O. Louro. 2011. Exploration of the ‘cytochromome’ of Desulfuromonas acetoxidans, a marine bacterium capable of powering microbial fuel cells. Metallomics 3:349-353.

17.) Lovley, D.R. 2006. Microbial fuel cells: novel microbial physiologies and engineering approaches. Curr. Opin. Biotechnol. 17:327-332.

18.) Xuan, J., X.-D. Jia, L.-P. Jiang, E.S. Abdel-Halim, and J.-J. Zhu. 2012. Gold nanoparticle-assembled capsules and their application as hydrogen peroxide biosensor based on hemoglobin. Bioelectrochemistry 84:32-37.

19.) Schulz, H., R.A. Fabianek, E.C. Pellicioli, H. Hennecke, and L. Thony-Meyer. 1999. Heme transfer to the heme chaperone CcmE during cytochrome c maturation requires the CcmC protein, which may function independently of the ABC-transporter CcmAB. Proc. Natl. Acad. Sci. USA 96:6462-6467.

20.) Stevens, J.M., D.A. Mavridou, R. Hamer, P. Kritsiligkou, A.D. Goddard, and S.J. Ferguson. 2011. Cytochrome c biogenesis system. I. FEBS J. 278:4170-4178.

21.) Feissner, R.E., C.L. Richard-Fogal, E.R. Frawley, J.A. Loughman, K.W. Earley, and R.G. Kranz. 2006. Recombinant cytochromes c biogenesis systems I and II and analysis of haem delivery pathways in Escherichia coli. Mol. Microbiol. 60:563-577.

22.) Kranz, R.G., C. Richard-Fogal, J.S. Taylor, and E.R. Frawley. 2009. Cytochrome c biogenesis: mechanisms for covalent modifications and trafficking of heme and for heme-iron redox control. Microbiol. Mol. Biol. Rev. 73:510-528.

23.) Arslan, E., H. Schulz, R. Zufferey, P. Kunzler, and L. Thony-Meyer. 1998. Overproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichia coli. Biochem. Biophys. Res. Commun. 251:744-747.

24.) Londer, Y.Y. 2011. Expression of recombinant cytochromes c in E. coli. Methods Mol. Biol. 705:123-150.

25.) Ozawa, K., A.I. Tsapin, K.H. Nealson, M.A. Cusanovich, and H. Akutsu. 2000. Expression of a tetraheme protein, Desulfovibrio vulgaris Miyazaki F cytochrome c3, in Shewanella oneidensis. MR-1. Appl. Environ. Microbiol. 66:4168-4171.

26.) Shi, L., J.T. Lin, L.M. Markillie, T.C. Squier, and B.S. Hooker. 2005. Overexpression of multi-heme c-type cytochromes. BioTechniques 38:297-299.

27.) Pollock, W.B., M. Loutfi, M. Bruschi, B.J. Rapp-Giles, J.D. Wall, and G. Voordouw. 1991. Cloning, sequencing, and expression of the gene encoding the high-molecular-weight cytochrome c from Desulfovibrio vulgaris Hildenborough. J. Bacteriol. 173:220-228.

28.) Kern, M., and J. Simon. 2011. Production of recombinant multiheme cytochromes c in Wolinella succinogenes. Methods Enzymol. 486:429-446.

29.) Park, I., and B.C. Kim. 2011. Homologous overexpression of omcZ, a gene for an outer surface c-type cytochrome of Geobacter sulfurreducens by single-step gene replacement. Biotechnol. Lett. 33:2043-2048.

30.) Woodward, J.J., N.I. Martin, and M.A. Marletta. 2007. An Escherichia coli expression-based method for heme substitution. Nat. Methods 4:43-45.

31.) Schiött, T., M. Throne-Holst, and L. Hederstedt. 1997. Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis. J. Bacteriol. 179:4523-4529.

32.) Rivera, M., and F.A. Walker. 1995. Biosynthetic preparation of isotopically labeled heme. Anal. Biochem. 230:295-302.

33.) Bryson, D., P.L. Lim, A. Lawson, S. Manjunath, and G.M. Raner. 2011. Isotopic labeling of the heme cofactor in cytochrome P450 and other heme proteins. Biotechnol. Lett. 33:2019-2026.

34.) Chen, W., C.S. Russell, Y. Murooka, and S.D. Cosloy. 1994. 5-Aminolevulinic acid synthesis in Escherichia coli requires expression of hemA. J. Bacteriol. 176:2743-2746.

35.) Bunce, R.A., C.L. Schilling, and M. Rivera. 1997. Synthesis of [1,2-13C]- and [2,3-13C]-labeled δ-aminolevulinic acid. J. Labelled Comp. Radiopharm. 39:669-675.

36.) Rivera, M., and G.A. Caignan. 2004. Recent developments in the 13C NMR spectroscopic analysis of paramagnetic hemes and heme proteins. Anal. Bioanal. Chem. 378:1464-1483.

37.) Datsenko, K.A., and B.L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645.

38.) Qian, Y., C.M. Paquete, R.O. Louro, D.E. Ross, E. Labelle, D.R. Bond, and M. Tien. 2011. Mapping the iron binding site(s) on the small tetraheme cytochrome of Shewanella oneidensis MR-1. Biochemistry 50:6217-6224.

39.) Pitts, K.E., P.S. Dobbin, F. Reyes-Ramirez, A.J. Thomson, D.J. Richardson, and H.E. Seward. 2003. Characterization of the Shewanella oneidensis MR-1 decaheme cytochrome MtrA: expression in Escherichia coli confers the ability to reduce soluble Fe(III) chelates. J. Biol. Chem. 278:27758-27765.

40.) Fernandes, A.P., I. Couto, L. Morgado, Y.Y. Londer, and C.A. Salgueiro. 2008. Isotopic labeling of c-type multiheme cytochromes overexpressed in E. coli. Protein Expr. Purif. 59:182-188.

41.) Studier, F.W. 2005. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41:207-234.

42.) Harnois, T., M. Rousselot, H. Rogniaux, and F. Zal. 2009. High-level production of recombinant Arenicola marina globin chains in Escherichia coli: a new generation of blood substitute. Artif. Cells Blood Substit. Immobil. Biotechnol. 37:106-116.

43.) Francis, R.T., and R.R. Becker. 1984. Specific indication of hemoproteins in polyacrylamide gels using a double-staining process. Anal. Biochem. 136:509-514.

44.) Druyan, R., B. Debernar, and M. Rabinowi. 1969. Turnover of cytochromes labeled with delta-aminolevulinic acid-3H in rat liver. J. Biol. Chem. 244:5874-5878.

45.) von Wachenfeldt, C., and L. Hederstedt. 1990. Bacillus subtilis 13-kilodalton cytochrome c-550 encoded by cccA consists of a membrane-anchor and a heme domain. J. Biol. Chem. 265:13939-13948.

46.) Londer, Y.Y., P.R. Pokkuluri, J. Erickson, V. Orshonsky, and M. Schiffer. 2005. Heterologous expression of hexaheme fragments of a multidomain cytochrome from Geobacter sulfurreducens representing a novel class of cytochromes c. Protein Expr. Purif. 39:254-260.

47.) Louro, R.O., I.J. Correia, L. Brennan, I.B. Coutinho, A.V. Xavier, and D.L. Turner. 1998. Electronic structure of low-spin ferric porphyrins: 13C NMR studies of the influence of axial ligand orientation. J. Am. Chem. Soc. 120:13240-13247.

48.) Salgueiro, C.A., D.L. Turner, H. Santos, J. LeGall, and A.V. Xavier. 1992. Assignment of the redox potentials to the four haems in Desulfovibrio vulgaris cytochrome c3 by 2D-NMR. FEBS Lett. 314:155-158.

49.) Bertini, I., I.C. Felli, R. Kummerle, D. Moskau, and R. Pierattelli. 2004. 13C-13C NOESY: an attractive alternative for studying large macromolecules. J. Am. Chem. Soc. 126:464-465.

50.) Erbil, W.K., M.S. Price, D.E. Wemmer, and M.A. Marletta. 2009. A structural basis for H-NOX signaling in Shewanella oneidensis by trapping a histidine kinase inhibitory conformation. Proc. Natl. Acad. Sci. USA 106:19753-19760.

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