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The octahaem MccA is a haem c–copper sulfite reductase

Nature volume 520, pages 706–709 (2015)Cite this article

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Abstract

The six-electron reduction of sulfite to sulfide is the pivot point of the biogeochemical cycle of the element sulfur1. The octahaem cytochrome c MccA (also known as SirA) catalyses this reaction for dissimilatory sulfite utilization by various bacteria. It is distinct from known sulfite reductases because it has a substantially higher catalytic activity and a relatively low reactivity towards nitrite. The mechanistic reasons for the increased efficiency of MccA remain to be elucidated. Here we show that anoxically purified MccA exhibited a 2- to 5.5-fold higher specific sulfite reductase activity than the enzyme isolated under oxic conditions2. We determined the three-dimensional structure of MccA to 2.2 Å resolution by single-wavelength anomalous dispersion. We find a homotrimer with an unprecedented fold and haem arrangement, as well as a haem bound to a CX15CH motif3. The heterobimetallic active-site haem 2 has a Cu(I) ion juxtaposed to a haem c at a Fe–Cu distance of 4.4 Å. While the combination of metals is reminiscent of respiratory haem–copper oxidases, the oxidation-labile Cu(I) centre of MccA did not seem to undergo a redox transition during catalysis. Intact MccA tightly bound SO2 at haem 2, a dehydration product of the substrate sulfite that was partially turned over due to photoreduction by X-ray irradiation, yielding the reaction intermediate SO. Our data show the biometal copper in a new context and function and provide a chemical rationale for the comparatively high catalytic activity of MccA.

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Figure 1: Structure of W. succinogenes MccA.
Figure 2: Haem–copper active sites in respiratory enzymes.
Figure 3: Sulfite reduction at haem 2 of MccA.
Figure 4: Mechanism of stepwise sulfite reduction by MccA.

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Protein Data Bank

Data deposits

Structural models and structure factors have been deposited in the Protein Data Bank under accession numbers 4RKM (form I) and 4RKN (form II).

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (EI 520/5 to O.E. and SI 848/7 to J.S.) and the BIOSS Centre for Biological Signaling Studies at Albert-Ludwigs-Universität Freiburg. We thank S. Andrade for assistance with stopped-flow spectrometry, P. Kroneck, T. Clarke and D. Richardson for stimulating discussions, and the beamline staff at the Swiss Light Source, Villigen, Switzerland, for assistance with data collection.

Author information

Authors and Affiliations

  1. Lehrstuhl Biochemie, Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Albertstrasse 21, 79104 Freiburg, Germany,

    Bianca Hermann & Oliver Einsle

  2. Department of Biology, Microbial Energy Conversion & Biotechnology, Technische Universität Darmstadt, Schnittspahnstrasse 10, 64287 Darmstadt, Germany,

    Melanie Kern, Luigi La Pietra & Jörg Simon

  3. BIOSS Centre for Biological Signalling Studies, Schänzlestrasse 1, 79104 Freiburg, Germany,

    Oliver Einsle

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  1. Bianca Hermann
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Contributions

B.H., M.K. and L.L.P. performed the experiments, B.H. and O.E. built and refined the structural model, all authors designed the experiments, B.H., O.E. and J.S. wrote the manuscript.

Corresponding authors

Correspondence to Jörg Simon or Oliver Einsle.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Comparison of monomer structure, haem packing and quaternary structure in multihaem c enzymes.

a–e, Monomer structure is shown on the left, haem packing in the centre, and quaternary structure on the right. a, W. succinogenes sulfite reductase MccA. b, W. succinogenes nitrite reductase NrfA (PDB accession 1FS7)10. c, N. europaea hydroxylamine oxidoreductase (PDB accession 4FAS)33. d, Hexameric T. nitratireducens nitrite reductase (PDB accession 3SXQ)15. e, S. oneidensis tetrathionate reductase (PDB accession 1SP3). All haem arrangements are superimposed onto that of MccA (red).

Extended Data Figure 2 Uncommon structural features of MccA.

Haem group 8 is linked to an unusual CX15CH motif that results in an extended loop region between C562 and C578 (red), dislocating haem 8 from the regular, perpendicular haem-packing pattern otherwise observed in multihaem enzymes. This region is adjacent to a loop starting at the copper ligand C495 that contains the only non-proline cis-peptide bond of MccA between G508 and F509. Conceivably, the action of the peptidyl isomerase MccB is required to generate this conformation after insertion of Cu(I), since the deletion of the mccB gene prevented the formation of stable MccA2.

Extended Data Figure 3 Identification of the additional heteroatom as copper based on anomalous difference electron density maps.

a, Diffraction data sets were collected at the absorption edges of Co (7,710 eV), Ni (8,333 eV), Cu (8,974 eV), Zn (9,664 eV) and As (11,866 eV, not shown in plot). b, Integrated electron density is shown for a sphere with r = 1.0 Å around the Fe positions (grey) and the heterometal site (green) indicated in the individual data sets. The heterometal absorption properties behave as expected for Cu. Error bars represent standard deviations for data from 12 individual observations in the asymmetric unit of the crystal form. c, Anomalous difference (F+− F−) electron density maps calculated for the individual data sets at the elemental absorption edges and contoured at the 5σ level. A maximum is observed only at energies corresponding to Cu and above, allowing for an unambiguous identification.

Extended Data Figure 4 Structure of partially copper-depleted MccA.

Protein isolated under oxic conditions commonly showed a decreased copper content of 0.2–0.5 Cu per monomer. The electron density surrounding the active site was best interpreted as a mixture of copper-containing monomers, probably with bound SO2 as in Fig. 3a, and an oxidatively damaged, Cu-depleted form, where the thiol groups of C399 and C495 have rotated away from the active site to form a disulfide bond. This leads to improved access to the active site at the distal axial position of the iron atom of haem 2, allowing for the binding of SO32−. The short distance of 1.4 Å between a sulfite oxygen and the Cu(I) ion indicated that the two indeed represent alternative and mutually exclusive conformations, forms I and II, that overlay in the crystal.

Extended Data Figure 5 Representative steady-state kinetic analysis of MccA.

a, The reactivity for sulfite and nitrite was assayed by following the reoxidation of benzyl viologen. MccA form I, isolated in the strict absence of dioxygen, showed higher activities for sulfite (black) and nitrite (green) than form II, isolated under oxic conditions (sulfite, blue; nitrite, yellow). For sulfite reduction, the kinetic parameters obtained were vmax = 151.3 ± 0.5 U mg−1 and KM = 43.9 ± 2.3 µM for form I and vmax = 71.8 ± 0.5 U mg−1 and KM = 63.9 ± 9.4 µM for form II (see Extended Data Table 2 for parameters). b, pH dependence of the sulfite-reducing (black) and nitrite-reducing (green) activity of MccA. Sulfite reduction shows an apparent maximum at pH 6.2. All measurements were carried out in triplicate with the error bars representing standard deviations between individual experiments.

Extended Data Figure 6 Difference electron density maps for bound substrate and intermediates.

a, b, The stereo images show monomer D (a) and monomer H (b) of the P21 structure of form I MccA (Extended Data Table 1). Whereas in a an SO2 molecule is bound to the Fe ion of haem group 2 together with a single H2O molecule, photoreduction has led to the generation of a second H2O, leaving SO bound to haem. The quality of the electron density maps allowed us to discern S and O atoms, but the state depicted in b may represent either an S(II) = O adduct (Fig. 4b) or the further reduced S(0)–O (Fig. 4c). All maps are Fo − Fc omit difference electron density maps calculated in the absence of ligands.

Extended Data Figure 7 Copper detection by time-resolved electron excitation spectroscopy of W. succinogenes MccA.

a, Owing to the presence of eight haem groups, a hypothetical, calculated absorption band of a typical type I copper site with an extinction coefficient of approximately 5,000 M−1 cm−1 at 600 nm (grey) would be difficult to detect in a background of oxidized (black) or reduced (purple) haems, but may possibly be extracted from the well-characterized absorption of the porpyrin cofactors. b, Form I MccA as isolated shows all haem groups in the oxidized state, with copper present as Cu(I) (black trace). Reduction with 100-fold molar excess of Ti(III) citrate in a stopped-flow spectrophotometer rapidly leads to the emergence of a typical ferrohaem spectrum (purple). For oxidation of reduced protein see Extended Data Fig. 8. c, Kinetic traces at 552 nm (red) and 600 nm (blue) show that the rise of the α-band at 552 nm is not accompanied by a relevant change at 600 nm.

Extended Data Figure 8 Reoxidation of W. succinogenes MccA with sulfite.

MccA as isolated in the oxidized state (black) and subsequently fully reduced by Ti(III) (purple) to be reactive towards sulfite. After removal of excess reductant, the stepwise addition of sulfite to reduced MccA leads to successive oxidation of haem centres (inset left, grey spectra). However, even at a large excess of sulfite (250 mM) the oxidation of MccA is incomplete. Assuming nearly identical spectroscopic properties for all haem groups, the number of reduced haems can be deduced from the relative position of the α-band at 552 nm between the oxidized state (0 haems reduced, black) and the Ti(III)-reduced state (8 haems reduced, purple). This evaluation shows that sulfite only obtains up to 4 electrons from fully reduced MccA (inset right). As the enzyme forms a trimer with 24 haem groups, a total of 12 electrons are drawn from the system, sufficient to fully reduce 2 sulfite molecules to sulfide.

Extended Data Table 1 Data collection and refinement statistics
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Extended Data Table 2 Kinetic parameters for MccA
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Hermann, B., Kern, M., La Pietra, L. et al. The octahaem MccA is a haem c–copper sulfite reductase. Nature 520, 706–709 (2015). https://doi.org/10.1038/nature14109

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