PubAg

Main content area

Plasticity in the High Affinity Menaquinone Binding Site of the Cytochrome aa3-600 Menaquinol Oxidase from Bacillus subtilis

Author:
Yi, Sophia M., Taguchi, Alexander T., Samoilova, Rimma I., O’Malley, Patrick J., Gennis, Robert B., Dikanov, Sergei A.
Source:
Biochemistry 2015 v.54 no.32 pp. 5030-5044
ISSN:
1520-4995
Subject:
Bacillus subtilis, Escherichia coli, binding sites, catalytic activity, cytochrome c, electrochemistry, electron transfer, histidine, hydrogen bonding, membrane potential, menaquinones, mutants, mutation, nitrogen, protons
Abstract:
Cytochrome aa₃-600 is a terminal oxidase in the electron transport pathway that contributes to the electrochemical membrane potential by actively pumping protons. A notable feature of this enzyme complex is that it uses menaquinol as its electron donor instead of cytochrome c when it reduces dioxygen to water. The enzyme stabilizes a menasemiquinone radical (SQ) at a high affinity site that is important for catalysis. One of the residues that interacts with the semiquinone is Arg70. We have made the R70H mutant and have characterized the menasemiquinone radical by advanced X- and Q-band EPR. The bound SQ of the R70H mutant exhibits a strong isotropic hyperfine coupling (a₁₄N ≈ 2.0 MHz) with a hydrogen bonded nitrogen. This nitrogen originates from a histidine side chain, based on its quadrupole coupling constant, e²qQ/h = 1.44 MHz, typical for protonated imidazole nitrogens. In the wild-type cyt aa₃-600, the SQ is instead hydrogen bonded with Nε from the Arg70 side chain. Analysis of the ¹H 2D electron spin echo envelope modulation (ESEEM) spectra shows that the mutation also changes the number and strength of the hydrogen bonds between the SQ and the surrounding protein. Despite the alterations in the immediate environment of the SQ, the R70H mutant remains catalytically active. These findings are in contrast to the equivalent mutation in the close homologue, cytochrome bo₃ ubiquinol oxidase from Escherichia coli, where the R71H mutation eliminates function.
Agid:
5321967