Purification and spectroscopic characterization of Ctb, a group III truncated hemoglobin implicated in oxygen metabolism in the food-borne pathogen Campylobacter jejuni

Abstract

Campylobacter jejuni is a food-borne bacterial pathogen that possesses two distinct hemoglobins, encoded by the ctb and cgb genes. The former codes for a truncated hemoglobin (Ctb) in group III, an assemblage of uncharacterized globins in diverse clinically and technologically significant bacteria. Here, we show that Ctb purifies as a monomeric, predominantly oxygenated species. Optical spectra of ferric, ferrous, O(2)- and CO-bound forms resemble those of other hemoglobins. However, resonance Raman analysis shows Ctb to have an atypical nu(Fe)(-)(CO) stretching mode at 514 cm(-)(1), compared to those of the other truncated hemoglobins that have been characterized so far. This implies unique roles in ligand stabilization for TyrB10, HisE7, and TrpG8, residues highly conserved within group III truncated hemoglobins. Because C. jejuni is a microaerophile, and a ctb mutant exhibits O(2)-dependent growth defects, one of the hypothesized roles of Ctb is in the detoxification, sequestration, or transfer of O(2). The midpoint potential (E(h)) of Ctb was found to be -33 mV, but no evidence was obtained in vitro to support the hypothesis that Ctb is reducible by NADH or NADPH. This truncated hemoglobin may function in the facilitation of O(2) transfer to one of the terminal oxidases of C. jejuni or, instead, facilitate O(2) transfer to Cgb for NO detoxification.

FIGURE 1

Sequence alignment of C. jejuni trHb with various trHbs, Cgb and the Hmp heme domain. The sequence comparison was carried out using Clustal W. The top eight sequences are trHbs, followed by the non-truncated globin (Cgb) of C. jejuni and the N-terminal heme domain of E. coli flavohemoglobin (Hmp). The locations of the respective helices (A-H) are displayed in the bottom line below the aligned sequences. Conserved residues are shaded. Sequence data for various hemoglobins were obtained from TIGR.

FIGURE 2

Electronic absorbance spectra of pure Ctb. All spectra were scanned against a 50 mM Tris HCl pH 7.0 baseline and are normalised to heme concentration (0.001 nM). Native Ctb corresponds to the globin as it elutes from the gel filtration column (peak maxima at 411, 512, 542, 578 and 640 nm). The other forms of Ctb were generated as described in the text. Peak maxima are as follows. Fe²⁺, 432 and 565 nm; Fe³⁺, 410, 512, 542, 582 and 640 nm; Fe²⁺-CO, 421, 538 and 569 nm; Fe²⁺-O₂, 414, 542 and 578 nm.

FIGURE 3

Redox titration of Ctb. The electrochemical potential was plotted against % oxidised Ctb/ % reduced Ctb in a semilog arithmetic plot according to the Nernst equation. Reducing (diamonds, triangles) and oxidising (squares) titrations are shown. The line has been fitted to all three titrations (R² = 0.83).

FIGURE 4

Resonance Raman spectra of ferric Ctb. The Raman spectrum of the ferric form was obtained at pH 7.0, 9.5 and 10.5.

FIGURE 5

Top: Resonance Raman spectra of deoxy and CO-bound derivatives of Ctb. The Raman spectra of the ferrous deoxy form (top) and the Fe²⁺-CO form (bottom) of Ctb.

FIGURE 6

Inverse correlation diagram of the Fe-CO stretching frequency versus the C-O stretching frequency of various heme proteins, and comparison with Ctb. The solid inverted triangles represent trHbN in closed (trHbN_c, upper) and open (trHbN_o, lower) conformations. Nearby, the solid squares represent Hmp in closed and open conformations. TrHbO is represented by the red, solid circle in the top left portion of the plot, and Ctb by the circle in the centre of the L=His line. The striped circle (extreme top left), striped square and striped triangle (middle) represent horseradish peroxidase, cytochrome c peroxidase and Mb, respectively. The L=Cys line corresponds to those proteins where the proximal ligand to the heme is the thiolate side chain of cysteine, the L=His line corresponds to those proteins where the proximal ligand is the imidazole/imidazolate side chain of histidine, and the 5C line corresponds to those proteins where the CO-bound form is penta-coordinate.