Globins are an ancient and diverse superfamily of proteins. The globins of microorganisms were relatively ignored for many decades after their discovery by Warburg in the 1930s and rediscovery by Keilin in the 1950s. The relatively recent focus on them has been fuelled by recognition of their structural diversity and fine-tuning to fulfill (probably) discrete functions but particularly by the finding that a major role of certain globins is in protection from the stresses caused by exposure to nitric oxide (NO)--itself a molecule that has attracted intense curiosity recently. At least three classes of microbial globin are recognised, all having features of the classical globin protein fold. The first class is typified by the myoglobin-like haemprotein Vgb from the bacterium Vitreoscilla, which has attracted considerable attention because of its ability to improve growth and metabolism for biotechnological gain in a variety of host cells, even though its physiological function is not fully understood. The truncated globins are widely distributed in bacteria, microbial eukaryotes as well as plants and are characterised by being 20-40 residues shorter than Vgb. The polypeptide is folded into a two-over-two helical structure while retaining the essential features of the globin superfamily. Roles in oxygen and NO metabolism have been proposed. The third and best understood class comprises the flavohaemoglobins, which were first discovered and partly characterised in yeast. These are distinguished by the presence of an additional domain with binding sites for FAD and NAD(P)H. Widely distributed in bacteria, these proteins undoubtedly confer protection from NO and nitrosative stresses, probably by direct consumption of NO. However, a bewildering array of enzymatic capabilities and the presence of an active site in the haem pocket reminiscent of peroxidases hint at other functions. A full understanding of microbial globins promises advances in controlling the interactions of pathogenic bacteria with their animal and plant hosts, and manipulations of microbial oxygen transfer with biotechnological applications.

The microaerophilic bacterium Campylobacter jejuni is a significant food-borne pathogen and is predicted to possess two terminal respiratory oxidases with unknown properties. Inspection of the genome reveals an operon (cydAB) apparently encoding a cytochrome bd-like oxidase homologous to oxidases in Escherichia coli and Azotobacter vinelandii. However, C. jejuni cells lacked all spectral signals characteristic of the high-spin hemes b and d of these oxidases. Mutation of the cydAB operon of C. jejuni did not have a significant effect on growth, but the mutation reduced formate respiration and the viability of cells cultured in 5% oxygen. Since cyanide resistance of respiration was diminished in the mutant, we propose that C. jejuni CydAB be renamed CioAB (cyanide-insensitive oxidase), as in Pseudomonas aeruginosa. We measured the oxygen affinity of each oxidase, using a highly sensitive assay that exploits globin deoxygenation during respiration-catalyzed oxygen uptake. The CioAB-type oxidase exhibited a relatively low affinity for oxygen (K m ‫؍‬ 0.8 M) and a V max of >20 nmol/mg/s. Expression of cioAB was elevated fivefold in cells grown at higher rates of oxygen provision. The alternative, ccoNOQP-encoded cyanide-sensitive oxidase, expected to encode a cytochrome cb-type enzyme, plays a major role in the microaerobic respiration of C. jejuni, since it appeared to be essential for viability and exhibited a much higher oxygen affinity, with a K m value of 40 nM and a V max of 6 to 9 nmol/mg/s. Lowtemperature photodissociation spectrophotometry revealed that neither oxidase has ligand-binding activity typical of the heme-copper oxidase family. These data are consistent with cytochrome oxidation during photolysis at low temperatures.

Campylobacter jejuni contains two hemoglobins, Cgb and Ctb. Cgb has been suggested to perform an NO detoxification reaction to protect the bacterium against NO attack. On the other hand, the physiological function of Ctb, a class III truncated hemoglobin, remains unclear. By using CO as a structural probe, resonance Raman data show that the distal heme pocket of Ctb exhibits a positive electrostatic potential. In addition, two ligand-related vibrational modes, Fe-O 2 and O-O , were identified in the oxy derivative, with frequencies at 542 and 1132 cm ؊1 , respectively, suggesting the presence of an intertwined H-bonding network surrounding the heme-bound ligand, which accounts for its unusually high oxygen affinity (222 M ؊1 ). Mutagenesis studies of various distal mutants suggest that the heme-bound dioxygen is stabilized by H-bonds donated from the Tyr(B10) and Trp(G8) residues, which are highly conserved in the class III truncated hemoglobins; furthermore, an additional H-bond donated from the His(E7) to the Tyr(B10) further regulates these H-bonding interactions by restricting the conformational freedom of the phenolic side chain of the Tyr(B10). Taken together, the data suggest that it is the intricate balance of the H-bonding interactions that determines the unique ligand binding properties of Ctb. The extremely high oxygen affinity of Ctb makes it unlikely to function as an oxygen transporter; on the other hand, the distal heme environment of Ctb is surprisingly similar to that of cytochrome c peroxidase, suggesting a role of Ctb in performing a peroxidase or P450-type of oxygen chemistry.

Summary Consistent with its role as a nitric oxide (NO)-detoxifying globin inCampylobacter jejuni , Cgb ( Campylobacter globin) expression is strongly and specifically induced following exposure to nitrosative stress, suggesting a previously unrecognized capacity for NOrelated stress sensing in this food-borne pathogen. In this study, Fur and PerR have been eliminated as major regulators of cgb , and NssR (Cj0466), a member of the Crp-Fnr superfamily, has been identified as the major positive regulatory factor that controls nitrosative stress-responsive expression of this gene. Accordingly, disruption of nssR resulted in the abolition of inducible cgb expression, which was restored by a complementing chromosomal insertion of the wild-type gene with its indigenous promoter at a second location. The NssR-deficient mutant was more sensitive to NO-related stress than a cgb mutant and this phenotype most likely arises from the failure of these cells to induce other NO-responsive components in addition to Cgb. Indeed, analysis of global gene expression, by microarray and confirmatory real-time polymerase chain reaction (PCR) in the wild type and nssR mutant, not only confirmed the dependence of inducible cgb expression on NssR, but also revealed for the first time a novel NssR-dependent nitrosative stress-responsive regulon. This regulon of at least four genes includes Cj0465c, a truncated globin. Consistent with NssR being a Crp-Fnr superfamily member, an Fnr-like binding sequence (TTAAC-N 4 -GTTAA) was found upstream of each gene at locations ----40.5 to ----42.5 relative to the centre of the binding sites and the transcription start point. Sitedirected mutagenesis confirmed that this cis -acting motif mediates the nitrosative stress-inducible expression of cgb .

Of the three groups of haemoglobins identified in micro-organisms (single-domain globins, flavohaemoglobins and truncated globins), the last group is the least well understood. The function of the truncated haemoglobin (Ctb) encoded by Cj0465c in the microaerophilic food-borne bacterial pathogen Campylobacter jejuni was investigated by constructing a ctb mutant and characterizing its phenotype. The effects of the ctb mutation on the kinetics of terminal oxidase function in C. jejuni were investigated using oxyleghaemoglobin and oxymyoglobin as sensitive reporters of O 2 consumption. The V max of ctb mutant cells for O 2 , calculated using either globin, was greater than that of wild-type cells at extracellular O 2 concentrations up to~1 mM, suggesting a role for Ctb in moderating O 2 supply for reduction by high-affinity terminal oxidases. However, cells mutated in ctb were disadvantaged when grown under conditions of high aeration, as revealed by measurements of growth yields and rates in batch culture. Furthermore, the rate at which ctb mutant cells consumed O 2 in an O 2 electrode (10-200 mM O 2 ) was approximately half the rate displayed by wild-type cells, reflecting a role for Ctb in respiration at physiologically relevant external O 2 concentrations. However, a lack of sensitivity of the mutant to paraquat or H 2 O 2 indicated that increased oxidative stress under such conditions was not the cause of these phenotypes. O 2 affinities of cells (K m values of approximately 40 nM and 1 mM) were unaffected by mutation of either Ctb or the full-length C. jejuni globin, Cgb. Although the gene encoding Ctb was found to be upregulated by S-nitrosoglutathione (GSNO) and the NO-donating compound S-nitroso-N-acetylpenicillamine (SNAP), a ctb mutant did not display sensitivity to a number of nitrosative stress-generating compounds. The authors conclude that Ctb is involved in moderating O 2 flux within C. jejuni.