Structural Characterization of Procaryotic Metal Reductase Systems
Final Report Abstract
Within the present project we have characterized a variety of novel c-type cytochromes in terms of their biophysical properties, three-dimensional structure and enzymatic activity. Starting out with gene products proposed to be involved in dissimilatory metal reduction we have studied the monoheme cytochrome OmcF, a member of the cytochrome c6 family, that serves as an electron carrier in the periplasm of the metal reducer Geobacter sulfurreducens and is likely reduced by an unusual cytochrome b6c quinol oxidase complex in the cytoplasmic membrane. In contrast, the ‘metal-reduction associated cytochrome’ MacA from the same organism, proposed as a central electron transfer hub at exactly this position, was analyzed and found to be a second di-heme peroxidase next to the canonical CcpA protein. We have produced and studied both proteins and several variants, and we have obtained a multitude of new information on this widespread family of bacterial oxygen detoxification enzymes. In particular we have been able to address the complex activation mechanism of the peroxidases, in which the reduction of a highpotential heme group by an external electron donor triggers the conformational rearrangement of three loop regions that eventually opens up access to the active site for the substrate. This mechanism is crucial to avoid the generation and release of reactive oxygen species. Through collaboration with Prof. Johannes Gescher, KIT Karlsruhe, we could also determine the structure of the CcpA ortholog from another metal reducer, Shewanella oneidensis, and show that here, too, it acts in the detoxification of reactive oxygen species rather than in electron transfer for metal reduction. A direct extension of the work on diheme peroxidases originated from the observation that a select few genes encoding such proteins contained an additional domain with yet another CXXCH motif for covalent heme attachment. In the genome sequence of Escherichia coli the open reading frame designated yhjA was found to encode a previously undetected cytochrome of this type, and we have consequently established the production and purification by recombinant expression. The solution of the threedimensional structure of YhjA was only possible after controlled dehydration of the crystal, and it revealed an arrangement of domains that indicated that the N-terminal fusion provides a very good model for the electron transfer complex of other CcpA proteins with their physiological redox partners. While hydrogen peroxide is a substrate of YhjA, it is reduced by the enzyme with low efficiency, indicating that the physiological substrate might be a different peroxide species. The theme of diheme peroxidases was found in a further variation in the dioxygenase RoxA from Xanthomonas sp., for which we solved a three-dimensional structure in collaboration with Prof. Dieter Jendrossek, Stuttgart. This extracellular enzyme cleaves polyisoprene chains, the main constituent of latex, natural rubber. While it catalyzes an almost entirely different chemistry, its heme arrangement and topology are directly derived from CcpA peroxidases, although the evolutionary extension of multiple loop regions has made the protein almost twice as large as the peroxidases. Our structure showed that the enzyme must follow a processive mechanism, but that it most likely does not undergo redox-driven conformational changes similar to the peroxidases. In summary, our work showed that MacA does not play the proposed role in metal reduction of G. sulfurreducens, but it has led to an extensive study into the mechanisms of CcpA peroxidases and the related enzymes YhjA and RoxA that sharpened our picture of the functionality and interplay of heme groups in this class of proteins.
Publications
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(2008). Crystal packing of the c6-type cytochrome OmcF from Geobacter sulfurreducens is mediated by an N-terminal Strep-tag II.
Acta Crystallogr. D64, 919-926
Lukat, P., Hoffmann, M. & Einsle, O.
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(2008). Crystallization of the extracellular rubber oxygenase RoxA from Xanthomonas sp. Strain 35Y.
Acta Crystallogr. F64, 123-125
Hoffmann, M., Braaz, R., Jendrossek, D. & Einsle, O.
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(2009). CcpA from Geobacter sulfurreducens is a Basic Di-heme Cytochrome c Peroxidase. J. Mol. Biol. 393, 951-965
Hoffmann, M., Seidel, J. & Einsle, O.
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(2011). Geobacter sulfurreducens cytochrome c peroxidases: Eletrochemical classifications of catalytic mechanisms. Biochemistry 50, 4513-4520
Ellis, K.E., Seidel, J., Einsle, O. & Elliott, S.J.
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(2011). Investigation of the electron transport chain to and the catalytic activity of the diheme cytochrome c peroxidase CcpA of Shewanella oneidensis. Appl. Environ. Microbiol. 77, 6172-6180
Schütz, B., Seidel, J., Sturm, G., Einsle, O. & Gescher, J.
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(2011). Structure and function of formate-dependent cytochrome c nitrite reductase. Meth. Enzymol. 496, 399-422
Einsle, O.
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(2012). Enzyme or Electrode? Structure 20, 1132-1134
Einsle, O.
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(2012). MacA is a second cytochrome c peroxidase in Geobacter sulfurreducens. Biochemistry 51, 2747-2456
Seidel, J., Hoffmann, M., Ellis, K.E., Seidel, A., Spatzal, T., Gerhardt, S., Elliott, S.J. & Einsle, O.
