Since its discovery, it has been established that the SHP gene occurs in over 42 species of divergent proteobacteria. Nevertheless, before initiation of this study, the protein had only been observed in one out of about 16 species of non-sulfur purple bacteria that had been examined, i.e. in Rb. sphaeroides, and thus appeared to be an anomaly. We have now shown that SHP is not confined to one or a few strains of Rb. sphaeroides, but is present in 9 out of 10 strains studied to date and the one negative strain, 2.4.3, is likely to be a separate species called Rb. azotoformans. Thus, the presence of SHP is not due to an isolated occurrence of gene transfer from some other species, but is truly characteristic of Rb. sphaeroides. If SHP had resulted from gene transfer, it may not have been functional or important to the survival of the species and absent from at least some strains. We now know that is not likely to be the case and the fact that the SHP operon is found in five related species of Rhodobacteraceae suggests that it is functional and of selective advantage to these species. Not only is the SHP operon present in other species of Rhodobacteraceae, but the relationships shown in Figures 2, 3 and 4 indicate that the SHPs and DHCs are more closely related to one another than to any other species, also indicating that there has been no recent gene transfer from outside the family. Our studies furthermore show that DHC was duplicated in the Rhodobacteraceae prior to speciation. It is impossible to say at this point how many times DHC was duplicated since there are species that have several copies of SHP and of sDHC. Gene duplication thus appears to be relatively common in the SHP operon.
We have shown that Rb. sphaeroides, Rb. changlensis, Rv. marinum, and presumably other Rhodobacteraceae (in the alpha class) have the CytB/mDHC chimera as part of the SHP operon. The only other species with a similar chimera are Rf. ferrireducens (in the beta class) and Cb. fetus, Ab. butzleri, and Wo. succinogenes (in the epsilon class). The latter three species are related, but all four have significant differences to one another and to Rb. sphaeroides such as the position of the SHP gene before the CytB (Figure 5). Furthermore, Wo. succinogenes does not have an sDHC, Cb. fetus and Ab. butzleri have regulatory genes as part of the operon, and none of these three SHPs have the C89-C97 disulfide. In terms of percentage identity, the Campylobacter and two related SHP operons are among the most divergent as shown in Figures 2 and 3. It is therefore likely that the chimera does not have a single origin.
Although evolution and gene transfer are important, we were particularly interested in what comparative analysis could tell us about the function of SHP in photosynthetic bacteria. One of the first clues to the function of SHP is the rare occurrence of ligand-switching upon reduction followed by oxygen binding. SHP is the only c-type heme protein known to bind oxygen [1, 2, 6, 9]. Among the b-type heme proteins, only the globins and cytochrome P-450 plus a few heme-containing sensor kinases and diguanylate cyclases form stable oxygen complexes, and in these cases, it is functionally important. By analogy to P-450, SHP can be postulated to be an oxygenase, which hydroxylates an unknown substrate. SHP has in fact been reported to oxidize nitric oxide to nitrate  as has been observed with other oxygen-binding heme proteins . Only Ac. vinosum  and Rb. sphaeroides  SHP have been tested and shown to bind oxygen, which is consistent with the lack of significant amino acid substitutions in the vicinity of the heme. On the other hand, Rs. rubrum and Rs. centenum SHPs have an N88D substitution of the sixth heme ligand and W84R and W84H substitutions that presumably stabilize Asp 88, respectively. N88 and W84 are strongly correlated in other species and are closely situated at the distal side of the heme. In computer models, the H84 substitution is close enough to bind to the heme iron, raising the interesting question whether it actually does so. In any case, the amino acid substitutions may be sufficient to preclude oxygen binding and suggest a different functional role for the Rhodospirillaceae SHPs.
The second clue to the function of SHP comes from the equally rare occurrence of binding organic amines, among which are Tris, HEPES, bis-tris-propane, hydroxylamine, and taurine . It is unlikely that the heme iron simultaneously binds both oxygen and organic amines, but there could be a separate binding site for co-substrate near the heme-bound oxygen which is rather open and exposed to solvent .
The third clue to the function of SHP comes from the genetic context of the SHP gene. SHP is in fact part of a three-gene operon along with a CytB/mDHC chimera and an sDHC. That is not only true for Rb. sphaeroides and the Rhodobacteraceae, but is generally true for the majority of SHP species as shown in Figure 5. The membrane-spanning cytochrome b has binding sites for two hemes and is related to cytochromes b that interact with quinones such as the membrane subunit of formate dehydrogenase . It is thus likely that a quinol reduces the CytB which reduces the mDHC and the sDHC, which then reduces SHP. The latter reaction has been clearly demonstrated for Rb. sphaeroides . That the diheme cytochromes could provide both necessary electrons to SHP to activate oxygen, is consistent with SHP functioning as an oxygenase.
The fourth clue to the function of SHP also comes from the genetic context. The majority of SHP operons include sensor kinase and response regulatory (SK/RR) genes with the exception of the three Rb. sphaeroides strains and Rhodobacter SW2 for which there are whole genome sequences and a few others as shown in Figure 5. There are three types of sensors associated with the SHP operons (Figure 1), which we will describe below. Of these three, the first two (SK1 and SK2) are equally common (74 and 60 examples out of ca 700 species known to date) and the third (SK3) rare (Arcobacter butzleri and Campylobacter fetus, Figure 1). The first two occur both within the SHP operon (15 and 12 examples) and in other contexts. Only SK1 is associated with SHP in the two photosynthetic families, Rhodospirillaceae and Chromatiaceae.
The first sensor kinase (SK1) and the accessory protein (orf1) are strongly correlated, both in the SHP operons and in other contexts in which they are found. Vibrio species are the only pathogens of which we are aware that have the SK1 sensor kinase. The SK1 sensor can be recognized by variations of the SGVYWQ and RSRSLWD motifs in the periplasmic domain. There are SK1 (RSP3512) and Orf1 (RSP3510) genes in Rb. sphaeroides strain 2.4.1 and they are located on chromosome II. On the other hand, SHP (RSP2021) is on chromosome I, but there is no known functional relationship. However, the presence of regulatory genes as part of the SHP operon in most species and the association of Rhodospirillaceae and Chromatiaceae SHPs with SK1 suggest that at least some of the Rhodobacteraceae SHPs may be regulated in part by SK1 as well. Since this study was initiated, the genome of Rhodobacter SW2 has been determined (Joint Genome Institute). These results show that while PrrBA genes are present, there are no regulatory genes associated with SHP, and SK1 and SK2 are absent.
The SK2 sensor is related to the enterohemorrhagic E. coli QseC quorum sensor that responds to autoinducer 3 (AI3, structure unknown) and also responds to epinephrine and norepinephrine from the host , both of which are organic amines. The QseC homologs include the important pathogens: Actinobacillus, Bordetella, Escherichia, Haemophilus, Legionella, Pasteurella, Pseudomonas, Salmonella, Vibrio, and Yersinia. Several of the pathogenic Burkholderia species (5 out of ca 14) contain the SHP operon including the SK2 sensor kinase. Presumably, these pathogenic bacteria respond to the concentration of AI3 excreted into the environment or to epinephrine produced by the host to activate virulence genes. Either the SHP operon is one of several that may be regulated by quorum sensing or may be turned on specifically to inactivate the autoinducer produced by competitors. Although there is an SK2 gene in Rb. sphaeroides, it is located elsewhere on the chromosome (RSP3431) and there is no known functional connection with SHP, yet its involvement in regulation of the Rb. sphaeroides SHP operon cannot be excluded at this time.
The third type of sensor kinase, SK3, is unique to Cb. fetus and Ab. butzleri and not found in Wo. succinogenes or Rb. sphaeroides or any other species of which we are aware. The more divergent SHP operon organization and the presence of a different SK indicate that the function of SHP is likely to be different in this group compared to the previous two, which may be regulated in part by SK1 and/or SK2.