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Identification of the nitrogen donor hydrogen bonded with the semiquinone at the Q(H) site of the cytochrome bo3 from Escherichia coli.

ABSTRACT: The selective (15)N isotope labeling was used for the identification of the nitrogen involved in a hydrogen bond formation with the semiquinone in the high-affinity Q(H) site in the cytochrome bo(3) ubiquinol oxidase. This nitrogen produces dominating contribution to X-Band (14)N ESEEM spectra. The 2D ESEEM (HYSCORE) experiments with the Q(H) site SQ in the series of selectively (15)N labeled bo(3) oxidase proteins have directly identified the N(epsilon) of R71 as an H-bond donor. In addition, selective (15)N labeling has allowed us for the first time to determine weak hyperfine couplings with the side-chain nitrogens from all residues around the SQ. Those are reflecting a distribution of the unpaired spin density over the protein in the SQ state of the quinone processing site.

SUBMITTER: Lin MT

PROVIDER: S-EPMC2645916 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Identification of the nitrogen donor hydrogen bonded with the semiquinone at the Q(H) site of the cytochrome bo3 from Escherichia coli.

Lin Myat T MT Samoilova Rimma I RI Gennis Robert B RB Dikanov Sergei A SA

Journal of the American Chemical Society 20081101 47

The selective (15)N isotope labeling was used for the identification of the nitrogen involved in a hydrogen bond formation with the semiquinone in the high-affinity Q(H) site in the cytochrome bo(3) ubiquinol oxidase. This nitrogen produces dominating contribution to X-Band (14)N ESEEM spectra. The 2D ESEEM (HYSCORE) experiments with the Q(H) site SQ in the series of selectively (15)N labeled bo(3) oxidase proteins have directly identified the N(epsilon) of R71 as an H-bond donor. In addition, s ...[more]

PMID: 18983149

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Project description:Selective (15)N isotope labeling of the cytochrome bo(3) ubiquinol oxidase from Escherichia coli with auxotrophs was used to characterize the hyperfine couplings with the side-chain nitrogens from residues R71, H98, and Q101 and peptide nitrogens from residues R71 and H98 around the semiquinone (SQ) at the high-affinity Q(H) site. The two-dimensional ESEEM (HYSCORE) data have directly identified N(?) of R71 as an H-bond donor carrying the largest amount of unpaired spin density. In addition, weaker hyperfine couplings with the side-chain nitrogens from all residues around the SQ were determined. These hyperfine couplings reflect a distribution of the unpaired spin density over the protein in the SQ state of the Q(H) site and the strength of interaction with different residues. The approach was extended to the virtually inactive D75H mutant, where the intermediate SQ is also stabilized. We found that N(?) of a histidine residue, presumably H75, carries most of the unpaired spin density instead of N(?) of R71, as in wild-type bo(3). However, the detailed characterization of the weakly coupled (15)N atoms from selective labeling of R71 and Q101 in D75H was precluded by overlap of the (15)N lines with the much stronger ~1.6 MHz line from the quadrupole triplet of the strongly coupled (14)N(?) atom of H75. Therefore, a reverse labeling approach, in which the enzyme was uniformly labeled except for selected amino acid types, was applied to probe the contribution of R71 and Q101 to the (15)N signals. Such labeling has shown only weak coupling with all nitrogens of R71 and Q101. We utilize density functional theory-based calculations to model the available information about (1)H, (15)N, and (13)C hyperfine couplings for the Q(H) site and to describe the protein-substrate interactions in both enzymes. In particular, we identify the factors responsible for the asymmetric distribution of the unpaired spin density and ponder the significance of this asymmetry to the quinone's electron transfer function.

Project description:Oxidized cytochrome bo reacts rapidly with micromolar concentrations of H2O2 to form a single derivative. The electronic absorption spectrum of this compound differs from that of the oxidized form of the enzyme reported by this laboratory [Watmough, Cheesman, Gennis, Greenwood and Thomson (1993) FEBS Lett. 319, 151-154]. It is characterized by a Soret maximum at 411 nm, increased absorbance at 555 nm, and reduced intensity at 624 nm. The apparent dissociation constant for this process is of the order of 4 x 10(-6) M, and the bimolecular rate constant for the formation of the new compound is (1.25-1.7) x 10(3) M-1.s-1. Electronic absorption difference spectroscopy shows this product to be identical with the compound formed from the reaction of the mixed-valence form of the enzyme with dioxygen. Investigation of this compound by room-temperature magnetic c.d. spectroscopy shows haem o to be neither high-spin nor low-spin ferric, but to have a spectrum characteristic of an oxyferryl species. There is no evidence for oxidation of the porphyrin ring. Therefore the binuclear centre of this species must consist of an oxyferryl haem (S = 1) coupled to a Cu(II) ion (S = 1/2) to form a new paramagnetic centre. The reaction was also followed by X-band e.p.r. spectroscopy, and this showed the disappearance in parallel with the formation of the oxyferryl species, of the broad g = 3.7, signal which arises from the weakly coupled binuclear centre in the oxidized enzyme. Since no new e.p.r.-detectable paramagnetic species were observed, the Cu(II) ion is presumed to be coupled to another paramagnet, possibly an organic radical. There is no evidence in the electronic absorption spectrum to indicate further reaction of cytochrome bo with H2O2 to form a second species. We argue that the circumstances of formation of this oxyferryl species are the same as those for the P form of cytochrome c oxidase, a species often regarded as containing a bound peroxide ion. The implications of these observations for the reaction mechanism of haem-copper terminal oxidases are discussed.

Dataset Information

Identification of the nitrogen donor hydrogen bonded with the semiquinone at the Q(H) site of the cytochrome bo3 from Escherichia coli.

Publications

Identification of the nitrogen donor hydrogen bonded with the semiquinone at the Q(H) site of the cytochrome bo3 from Escherichia coli.

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