TY - JOUR
T1 - Functional importance of Glutamate-445 and Glutamate-99 in proton-coupled electron transfer during oxygen reduction by cytochrome bd from Escherichia coli
AU - Murali, Ranjani
AU - Gennis, Robert B.
N1 - Publisher Copyright:
© 2018 Elsevier B.V.
PY - 2018/8
Y1 - 2018/8
N2 - The recent X-ray structure of the cytochrome bd respiratory oxygen reductase showed that two of the three heme components, heme d and heme b 595 , have glutamic acid as an axial ligand. No other native heme proteins are known to have glutamic acid axial ligands. In this work, site-directed mutagenesis is used to probe the roles of these glutamic acids, E445 and E99 in the E. coli enzyme. It is concluded that neither glutamate is a strong ligand to the heme Fe and they are not the major determinates of heme binding to the protein. Although very important, neither glutamate is absolutely essential for catalytic function. The close interactions between the three hemes in cyt bd result in highly cooperative properties. For example, mutation of E445, which is near heme d, has its greatest effects on the properties of heme b 595 and heme b 558 . It is concluded that 1) O 2 binds to the hydrophilic side of heme d and displaces E445; 2) E445 forms a salt bridge with R448 within the O 2 binding pocket, and both residues play a role to stabilize oxygenated states of heme d during catalysis; 3) E445 and E99 are each protonated accompanying electron transfer to heme d and heme b 595 , respectively; 4) All protons used to generate water within the heme d active site come from the cytoplasm and are delivered through a channel that must include internal water molecules to assist proton transfer: [cytoplasm] → E107 → E99 (heme b 595 ) → E445 (heme d) → oxygenated heme d.
AB - The recent X-ray structure of the cytochrome bd respiratory oxygen reductase showed that two of the three heme components, heme d and heme b 595 , have glutamic acid as an axial ligand. No other native heme proteins are known to have glutamic acid axial ligands. In this work, site-directed mutagenesis is used to probe the roles of these glutamic acids, E445 and E99 in the E. coli enzyme. It is concluded that neither glutamate is a strong ligand to the heme Fe and they are not the major determinates of heme binding to the protein. Although very important, neither glutamate is absolutely essential for catalytic function. The close interactions between the three hemes in cyt bd result in highly cooperative properties. For example, mutation of E445, which is near heme d, has its greatest effects on the properties of heme b 595 and heme b 558 . It is concluded that 1) O 2 binds to the hydrophilic side of heme d and displaces E445; 2) E445 forms a salt bridge with R448 within the O 2 binding pocket, and both residues play a role to stabilize oxygenated states of heme d during catalysis; 3) E445 and E99 are each protonated accompanying electron transfer to heme d and heme b 595 , respectively; 4) All protons used to generate water within the heme d active site come from the cytoplasm and are delivered through a channel that must include internal water molecules to assist proton transfer: [cytoplasm] → E107 → E99 (heme b 595 ) → E445 (heme d) → oxygenated heme d.
KW - Cytochrome bd
KW - Heme
KW - Proton transfer
KW - Respiration
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U2 - 10.1016/j.bbabio.2018.04.012
DO - 10.1016/j.bbabio.2018.04.012
M3 - Article
C2 - 29719208
AN - SCOPUS:85047073248
SN - 0005-2728
VL - 1859
SP - 577
EP - 590
JO - Biochimica et Biophysica Acta - Bioenergetics
JF - Biochimica et Biophysica Acta - Bioenergetics
IS - 8
ER -