Bovine liver catalase (BLC), catalase-related allene oxide synthase (cAOS) from Plexaura homomalla, and a recently isolated protein from the cattle pathogen Mycobacterium avium ssp. paratuberculosis (MAP-2744c (MAP)) are all tyrosinate-ligated heme enzymes whose crystal structures have been reported. cAOS and MAP have low (< 20%) sequence similarity to, and significantly different catalytic functions from, BLC. cAOS transforms 8R-hydroperoxy-eicosatetraenoic acid to an allene epoxide, whereas the MAP protein is an organic peroxide-dependent peroxidase. To shed light on the functional differences among these three proteins, we have investigated the heme iron coordination properties of these tyrosinate-ligated heme proteins in their ferric and ferrous states using magnetic circular dichroism and UV-visible absorption spectroscopy. The MAP protein shows remarkable spectral similarities to cAOS and BLC in its native Fe(III) state, but clear differences from ferric His93Tyr Mb, which may be attributed to the presence of an Arg+-Nω-H … O-Tyr (proximal heme axial ligand) hydrogen bond in the first three heme proteins. Furthermore, the spectra of Fe(III)-CN−, Fe(III)-NO, Fe(II)-NO (except for five-coordinate MAP), Fe(II)-CO, and Fe(II)-O2 states of cAOS and MAP, but not H93Y Mb, are also similar to the corresponding six-coordinate complexes of BLC, suggesting that a tyrosinate (Tyr-O−) is the heme axial ligand trans to the bound ligands in these complexes. The Arg+-Nω-H to −O-Tyr hydrogen bond would be expected to modulate the donor properties of the proximal tyrosinate oxyanion and, combined with the subtle differences in the catalytic site structures, affect the activities of cAOS, MAP and BLC.

Ligand binding and substitution reactions are important for metalloprotein folding and function. The heme sensor of a methyl-accepting chemotaxis GSU0935 is a c-type cytochrome from the bacterium Geobacter sulfurreducens. The heme domain switches one of its axial ligands from H(2)O to a low-spin ligand, presumably Met, upon reduction. The study analyzes the stability and folding kinetics of the ferric domain. Guanidine hydrochloride denaturation yields the low-spin heme species arising from coordination of the ferric heme by non-native His residues. The population of the low-spin species further increases and then declines during protein refolding. Kinetics and mutational effects suggest that His54, from the N-terminal region of the domain, is the transient ligand to the heme. The capture and release of a non-native ligand within the compact partially-folded structures illustrates the flexibility of the heme environment in GSU0935, which may relate to the domain sensor function.

The mutant hKv1.3_V388C channel shows inward current at potentials more negative than 100 mV when the central apore is closed (Pruetting et al., JBC 286:20031-20042). We believe that the V388C mutation in hKv1.3 channels opens a new pathway (s-pore) behind the K þ conducting apore. The entry of the spore from the extracellular side is presumably located at the backside of Y395 (Shaker position 445) and F384 (Shaker position 434). One spore seems to be formed between two neighboring S6 subunits and it runs parallel to the apore. For a more detailed characterization of the spore, we created a concatamer consisting of three hKv1.3_V388C asubunits linked together. Overexpression of this trimeric hKv1.3_V388C channel in COS7 cells yielded typical spore currents at potentials more negative than 100 mV similar to what was observed for the tetrameric hKv1.3_V388C channel. Electrophysiological properties of the trimeric and tetrameric channels were similar: currents at potentials more negative than 100 mV were not carried by protons or chloride ions and could not be reduced by peptide toxins (CTX, MTX) or TEA. The spore was mostly permeable to Na þ and Li þ . For the determination of the path of the spore in the hKv1.3_V388C channel we mutated single amino acids in the hKv1.3_V388C mutant channel that maybe involved in spore formation. Two of these mutant channels (hKv1.3_V388C_W384F, hKv1.3_V388C_Y395W) reduced current through the spore indicating that these positions might line the spore.