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

In the past three decades, our understanding of hemoglobins has grown to recognise new classes of proteins in addition to the classical vertebrate α- and β-globins, myoglobin and the symbiotic Hbs of leguminous plants. These ‘new’ globins - some of which may actually be amongst the oldest, or ancestral, globins in a phylogenetic context (1) - include the non-symbiotic plant Hbs, chimeric flavohemoglobins, and the trHbs. Globins may be classified functionally - into those that detoxify NO and those that are involved in O2 transfer or detoxification – or structurally into three categories (2). The first structural category contains flavoHbs that possess a C-terminal ferredoxin-NADP+ reductase-like domain and an N-terminal globin domain; bacterial (3, 4) and yeast (5) examples function in the enzymic removal of NO to produce nitrate (6, 7). The second category contains single domain Hbs that are very similar to the N-terminal domain of the flavoHbs; however, their functions appear more varied than those of the flavoHbs. As an example, Vgb, found in the obligate aerobe Vitreoscilla, is believed to function in the facilitation of O2 transfer to cytochrome bo’ (8), whereas the C. jejuni Hb, Cgb, confers tolerance to NO (9) and is up-regulated by NO and its congeners by an NO-activated transcriptional regulator (10). The trHbs comprise the third category: members possess an altered structure whereby the globin fold is edited from the three-over-three α-helical sandwich (3/3) characteristic of vertebrate globins to a two-over-two arrangement (11) and are thus sometimes called 2/2 Hbs (1). This results in a considerably smaller globin composed typically of 110-130 amino acids. A subdivision of the trHbs, based on the information available from more than 40 actual or putative trHb genes, was proposed by Wittenberg et al. (12). Three distinct groups were identified (I, II, III), with four subgroups occurring within group II. Few amino acids are strictly conserved throughout the trHb sequences, only the proximal HisF8 being invariant (see below). Note that we use here the terms group I, II and III in this context as defined by Wittenberg et al. (12) and not to distinguish between the truncated globins, myoglobin-like proteins and flavohemoglobins (13). The crystal structures of trHbs from Chlamydomonas, Paramecium (11), M. tuberculosis (14, 15) and Synechocystis (16) have been solved. The antiparallel helix pairs are comprised of B/E and G/H. The A helix is almost entirely deleted, the instability implied by this absence being remedied by the presence of a hydrophobic amino acid cluster in the AB region to allow efficient sealing of the proximal side of the heme pocket (11). There are three conserved Gly-Gly motifs located in, respectively, the AB hinge (11), the EF hinge and at the end of the pre-F loop (17) that are believed to stabilise the globin fold. Only group I and II trHbs possess the Gly-Gly motifs; little is known concerning the putative motifs that replace them in terms of stabilisation within group III. The F helix is attenuated to a single turn, the rest being replaced by an extended polypeptide segment termed the pre-F loop (11). Group II trHbs have a conserved EF loop, whereas this area in group I trHbs is shorter and lacking sequence conservation (18). The C helix is barely present and the CD-D region is reduced to approximately three residues, arguably the smallest polypeptide span that could join the C and E helices (12). There are only a few strictly conserved residues within the known trHb sequences. The trHbs without exception retain the conserved HisF8 residue as the proximal ligand to the heme. However, the conserved residues of the distal pocket are more variable. At the B9-B10 sites, there is a strongly conserved Phe-Tyr couplet, the TyrB10 being involved in heme ligand stabilisation (12). Position CD1 is conserved as Phe in group I and III trHbs; those residues in group II inhabiting the position can be Phe, Tyr or His. The E7 position is more variable: in group I trHbs the position is occupied mostly by GlnE7, group III trHbs invariably have HisE7, and in group II Ala, Ser or Thr can occupy this position. Phe almost always occupies the E14 position in all groups; this residue is believed to shield the heme from solvent in a role comparable to that of PheCD1 in other hemoglobins (12). The gram-negative microaerophilic bacterium Campylobacter jejuni is notable but not unique in possessing two different Hbs. In addition to Cgb, a single-domain (3/3) Hb (9), it possesses Ctb (19) that belongs to trHb group III. This is by far the least well-understood family of Hbs, despite the fact that it embraces globins (12) from the pathogens Bordatella pertussis and Mycobacterium avium and the obligately aerobic metal-leaching acidophilic bacterium Thiobacillus ferrooxidans. C. jejuni is now recognised as one of the most important causes of bacterial gastroenteritis worldwide (20). In humans, campylobacteriosis is mainly a food-borne disease. However, C. jejuni is commonly a gut commensal in many food-producing animals and birds and contamination of meat during processing is an important method of transfer (20). Cgb protects the bacterium from the toxic effects of NO (9), but the group III trHb has no clearly defined function. Its expression is, however, elevated in response to nitrosative stress, a response mediated by the NssR sensor/regulator (10). The microaerophilic nature of C. jejuni implies the presence of mechanisms for high affinity O2 binding and/or coping with O2 toxicity and it is possible that Ctb fulfils one of these functions (19). In the present work, we have cloned and over-expressed the C. jejuni Ctb (Cj0465c), characterized it through optical and resonance Raman spectroscopy, and determined its mid-point redox potential. This is the first detailed characterization of a group III trHb.