Involvement of Glutamic Acid 278 in the Redox Reaction of the Cytochrome c Oxidase from Paracoccus denitrificans Investigated by FTIR Spectroscopy

Abstract

The molecular processes concomitant with the redox reactions of wild-type and mutant cytochrome c oxidase from Paracoccus denitrificans were analyzed by a combination of protein electrochemistry and Fourier transform infrared (FTIR) difference spectroscopy. Oxidized-minus-reduced FTIR difference spectra in the mid-infrared (4000-1000 cm-1) reflecting full or stepwise oxidation and reduction of the respective cofactor(s) were obtained. In the 1800-1000 cm-1 range, these FTIR difference spectra reflect changes of the polypeptide backbone geometry in the amide I (ca. 1620-1680 cm-1) and amide II (ca. 1560-1540 cm-1) region in response to the redox transition of the cofactor(s). In addition, several modes in the 1600-1200 cm-1 range can be tentatively attributed to heme modes. A peak at 1746 cm-1 associated with the oxidized form and a peak at 1734 cm-1 associated with the reduced form were previously discussed by us as proton transfer between Asp or Glu side chain modes in the course of the redox reaction of the enzyme [Hellwig, P., Rost, B., Kaiser, U., Ostermeier, C., Michel, H., and Mäntele, W. (1996) FEBS Lett. 385, 53-57]. These signals were resolved into several components associated with the oxidation of different cofactors. For a stepwise potential titration from the fully reduced state (-0.5 V) to the fully oxidized state (+0.5 V), a small component at 1738 cm-1 develops in the potential range of approximately +0.15 V and disappears at more positive potentials while the main component at 1746 cm-1 appears in the range of approximately +0.20 V (all potentials quoted vs Ag/AgCl/3 M KCl). This observation clearly indicates two different ionizable residues involved in redox-induced proton transfer. The major component at 1746 cm-1 is completely lost in the FTIR difference spectra of the Glu 278 Gln mutant enzyme. In the spectrum of the subunit I Glu 278 Asp mutant enzyme, the major component of the discussed difference band is lost. In contrast, the complete difference signal of the wild-type enzyme is preserved in the Asp 124 Asn, Asp 124 Ser, and Asp 399 Asn variants, which are critical residues in the discussed proton pump channel as suggested from structure and mutagenesis experiments. On the basis of these difference spectra of mutants, we present further evidence that glutamic acid 278 in subunit I is a crucial residue for the redox reaction. Potential titrations performed simultaneously for the IR and for the UV/VIS indicate that the signal related to Glu 278 is coupled to the electron transfer to/from heme a; however, additional involvement of CuB electron transfer cannot be excluded.

A cooperative model for protonmotive heme‐copper oxidases. The role of heme a in the proton pump of cytochrome c oxidase

“…4) shows that the conserved E242 (facing a cavity delimitated by helices VI, II and X) can, on one side, establish a protonic connection, through protolytic residues of helix II and bound water molecules, with H61, ligand to the heme a Fe, or with protonatable groups of the heme a porphyrin. FTIR spectroscopy results indicate that the ionization state or the position of E278 (Pden) is coupled to electron transfer to/from heme a [43]. Mutation in Saccharomyces cerevisiae of the leucine corresponding to I66, located in the bovine oxidase between E242 and heme a (see Fig.…”

Redox-induced Protein Structural Changes in Cytochrome bo Revealed by Fourier Transform Infrared Spectroscopy and [13C]Tyr Labeling

“…Application of spectro-electrochemical cells (47)(48)(49)(50)(51)(52), attenuated total reflection FTIR apparatus (41,42,(53)(54)(55)(56), and a photochemical reduction technique (57-60) enabled us to examine protein structural changes upon reduction of the redox metal centers of respiratory terminal oxidases. Alternatively, photodissociation of CO from the reduced enzyme revealed the local structural change surrounding the heme-copper binuclear center of the E. coli cytochrome bo (61,62).…”

“…Alternatively, photodissociation of CO from the reduced enzyme revealed the local structural change surrounding the heme-copper binuclear center of the E. coli cytochrome bo (61,62). Previous studies using site-directed mutants revealed that a COOH group of Glu-286 at the end of the D-channel undergoes hydrogen bond changes upon full reduction of the metal centers (48,50,59,60) or CO photolysis of the reduced, CO-bound enzyme (61,62). In contrast, a lack of Cu B and the substitution of high spin heme o for heme b in the Tyr-288 mutants (63,64) obscured protein structural changes attributable to Tyr-288 at the end of the K-channel (59).…”

Electrochemical study of an electron shuttle diheme protein: The cytochrome c from T. thermophilus

“…A mixture of 19 mediators was added to the protein sample with a final concentration of 25 µM to accelerate the redox reaction [38]. A gold grid modified with a 1:1 aqueous solution of cysteamine and mercaptopropionic acid was used as working electrode, a platinum contact as counter electrode and an aqueous Ag/AgCl 3M KCl as reference electrode.…”

“…These relatively high wavenumbers confirm that the hemes are surrounded by a rather hydrophobic environment. The positive signal observed at 1717 cm -1 and 1720 cm -1 for cyt c550 [C] and cyt c552 can be attributed to an Asp or Glu residue [38,66,[69][70][71] which is protonated in the oxidized form and probably located in the hydrophobic environment surrounding the heme. Deprotonated propionates and acidic residues contribute between 1580 and 1520 cm -1 ((COO − ) as mode) and between 1480…”