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Axial Coordination of Heme in Ferric CcmE Chaperone Characterized by EPR Spectroscopy

Inés García-Rubio ^*, Martin Braun , Igor Gromov ^*, Linda Thöny-Meyer  and Arthur Schweiger ^*

^* Laboratorium für Physikalische Chemie and Institut für Mikrobiologie, ETH Zurich, 8093 Zurich, Switzerland

Correspondence: Address reprint requests to Inés García-Rubio, HCI F 233, Wolfgang-Pauli-Str. 10, 8093 Zurich, Switzerland. Tel.: 41-44-632-4445; Fax: 41-44-632-1021; E-mail: garciarubio{at}esr.phys.chem.ethz.ch.

In Escherichia coli cytochrome c maturation requires a set of eight proteins including the heme chaperone CcmE, which binds heme transiently, yet covalently. Several variants of CcmE were purified and analyzed by continuous-wave electron paramagnetic resonance, electron nuclear double resonance, and hyperfine sublevel correlation spectroscopy to investigate the heme axial coordination. Results reveal the presence of a number of coordination environments, two high-spin heme centers with different rhombicities, and at least one low-spin heme center. The low-spin species was shown to be an artifact induced by the presence of available histidines in the vicinity of the iron. Both of the high-spin forms are five-coordinated, and comparison of the spectra of the wild-type CcmE with those of the mutant CcmE^Y134H proves that the higher-rhombicity form is coordinated by Tyr¹³⁴. The low-rhombicity (axial) form does not have a histidine residue or a water molecule as an axial ligand. However, we identified exchangeable protons coupled to the iron ion. We propose that the axial form can be coordinated by a carboxyl group of an acidic residue in the flexible domain of the protein. The two species would represent two different conformations of the flexible -helix domain surrounding the heme. This conformational flexibility confers CcmE special dynamic properties that are certainly important for its function.