Functional importance of Glutamate-445 and Glutamate-99 in proton-coupled electron transfer during oxygen reduction by cytochrome bd from Escherichia coli

Abstract

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, 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 and heme b. It is concluded that 1) O binds to the hydrophilic side of heme d and displaces E445; 2) E445 forms a salt bridge with R448 within the O 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, 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) → E445 (heme d) → oxygenated heme d.

Homologous bd oxidases share the same architecture but differ in mechanism

“…1) as expected from mutagenesis and spectroscopy studies (Fig. 2) 8 . Heme d, however, found closer to the periplasmic side in the G. thermodenitrificans structure, and heme b 595 , oriented to the middle of the membrane 13 , occupy interchanged positions in E. coli as demonstrated by significantly better density fits (Figs.…”

“…2 and 3). The proposed heme arrangement is in line with the spectroscopic characterization of E. coli variants, in which Glu99 and Glu445, the axial ligands to heme b 595 and d, have been mutated 8,23–26 . In our model, heme b 595 is closer to the periplasmic space and ligated by Glu445 and heme d itself is positioned more to the middle of the membrane with Glu99 as axial ligand.…”

“…In our model, heme b 595 is closer to the periplasmic space and ligated by Glu445 and heme d itself is positioned more to the middle of the membrane with Glu99 as axial ligand. This is backed by the finding that mutating Glu445 indeed had the strongest effects on the ultraviolet (UV)–vis spectra of hemes b 595 and b 558 8 . Further on, it was shown that E. coli Glu445 is critical for the electrochemical properties of heme b 595 12,23 .…”

“…Further on, it was shown that E. coli Glu445 is critical for the electrochemical properties of heme b 595 12,23 . Mutations of Glu99 led to either the loss of the heme b 595 /heme d active site 24–26,33 , or solely to the loss of heme d 8,26 implying at least a proximity of Glu99 to heme d. We changed the non-conserved Glu74 A located at the surface to CydB to a phenylalanine residue. The residue is located at the level of heme d. The sequences of the mutagenic primers are listed in Supplementary Table 2.…”

“…In addition, they are required for a rapid dissociation of gaseous inhibitors, such as nitric oxide and enable growth under oxidative stress conditions in the presence of, e.g., H 2 O 2 6,7 . However, the mechanism of bd oxidases is still under debate due to lack of the structure of the Escherichia coli bd -I oxidase, one of the two bd oxidases in this bacterium that has been intensively used for most biochemical and biophysical characterizations 8 .…”