Search

Heme carrier HasA has a unique type of histidine/tyrosine heme iron ligation in which the iron ion is in a thermally driven two spin states equilibrium. We recently suggested that the H-bonding between Tyr75 and the invariantly conserved residue His83 modulates the strength of the iron-Tyr75 bond. To unravel the role of His83, we characterize the iron ligation and the electronic properties of both wild type and H83A mutant by a variety of spectroscopic techniques. Although His83 in wild type modulates the strength of the Tyr-iron bond, its removal causes detachment of the tyrosine ligand, thus giving rise to a series of pH-dependent equilibria among species with different axial ligation. The five coordinated species detected at physiological pH may represent a possible intermediate of the heme transfer mechanism to the receptor.

Sulfate-reducing bacteria contain a variety of multi-heme c-type cytochromes. The cytochrome of highest molecular weight (Hmc) contains 16 heme groups and is part of a transmembrane complex involved in the sulfate respiration pathway. We present the 2.42 A resolution crystal structure of the Desulfovibrio vulgaris Hildenborough cytochrome Hmc and a structural model of the complex with its physiological electron transfer partner, cytochrome c(3), obtained by NMR restrained soft-docking calculations. The Hmc is composed of three domains, which exist independently in different sulfate-reducing species, namely cytochrome c(3), cytochrome c(7), and Hcc. The complex involves the last heme at the C-terminal region of the V-shaped Hmc and heme 4 of cytochrome c(3), and represents an example for specific cytochrome-cytochrome interaction.

The protein HasA from the Gram negative bacteria Serratia marcescens is the first hemophore to be described at the molecular level. It participates to the shuttling of heme from hemoglobin to the outer membrane receptor HasR, which in turn releases it into the bacterium. HasR alone is also able to take up heme from hemoglobin but synergy with HasA increases the efficiency of the system by a factor of about 100. This iron acquisition system allows the bacteria to survive with hemoglobin as the sole iron source. Here we report the structures of a new crystal form of HasA diffracting up to 1.77A resolution as well as the refined structure of the trigonal crystal form diffracting to 3.2A resolution. The crystal structure of HasA at high resolution shows two possible orientations of the heme within the heme-binding pocket, which probably are functionally involved in the heme-iron acquisition process. The detailed analysis of the three known structures reveals the molecular basis regulating the relative affinity of the heme/hemophore complex.

Free iron availability is strongly limited in vertebrate hosts, making the iron acquisition by siderophores inappropriate. Pathogenic bacteria have developed various ways to use the host's iron from iron-containing proteins. Serratia marcescens can use the iron from hemoglobin through the secretion of a hemophore called HasA, which takes up the heme from hemoglobin and shuttles it to the receptor HasR, which in turn, releases heme into the bacterium. We report here the first crystal structure of such a hemophore, bound to a heme group at two different pH values and at a resolution of 1.9 A. The structure reveals a new original fold and suggests a hypothetical mechanism for both heme uptake and release.

The crystal structure of cytochrome c3 (M(r) 13,000) from Desulfovibrio desulfuricans (118 residues, four heme groups) has been crystallographically refined to 1.7 A resolution using a simulated annealing method, based on the structure-model at 2.5 A resolution, already published. The final R-factor for 10,549 reflections was 0.198 covering the range from 5.5 to 1.7 A resolution. The individual temperature factors were refined for a total of 1059 protein atoms, together with 126 bound solvent molecules. The structure has been analyzed with respect to its detailed conformational properties, secondary structure features, temperature factor behaviour, bound solvent sites and heme geometry and ligation. The characteristic secondary structures of the polypeptide chain of this molecule are one extended alpha-helix, a short beta-strand and 13 reverse turns. The four heme groups are located in different structural environments, all highly exposed to solvent. The particular structural features of the heme environments are compared to the four hemes of the cytochrome c3 from Desulfovibrio vulgaris Miyazaki.

The first three-dimensional structure of a dimeric, octa-heme cytochrome c3 (M(r) 26000) from Desulfovibrio desulfuricans Norway, established at 2.2 A resolution, is briefly presented and compared to the known 3-D-structures of different C3-type tetraheme cytochromes, in order to contribute to a better understanding of the function of multiheme clusters and of the role of conserved amino acids implicated in possible electron transfer pathways. The dimeric protein crystallizes in the space group P3(1)21 with a = 73.01 A, c = 61.81 A and the asymmetric unit contains one monomer subunit, the dimer being generated by the crystallographic two-fold axis. The 3-D-structure was solved using the molecular replacement method with a model based on the structure of the tetraheme cytochrome c3 (M(r) 13000) from D desulfuricans Norway, presently refined at 1.7 A resolution. The monomeric subunit has the same overall fold as all cytochromes c3 (M(r) 13000). Moreover, the heme core of all examined cytochromes c3 is highly conserved, but differences appear concerning the heme environments and the histidines, axial ligands of the heme-iron atoms.

The nature of the axial ligands of a heme group is an important factor in maintaining the oxidation-reduction potential of a c-type cytochrome. Cytochrome c3 from Desulfovibrio vulgaris Hildenborough contains four bis-histidinyl coordinated hemes with low oxidation-reduction potentials. Site-directed mutagenesis was used to generate a mutant in which histidine 70, the sixth axial ligand of heme 4, has been replaced by a methionine. The mutant protein was expressed in Desulfovibrio desulfuricans G200 at a level similar to the wild type cytochrome. A model for the three-dimensional structure of D. vulgaris Hildenborough cytochrome c3 was generated on the basis of the crystal structure of D. vulgaris Miyazaki cytochrome c3 in order to investigate the effects of the H70M mutation. The model, together with NMR data, suggested that methionine 70 has effectively replaced histidine 70 as the sixth axial ligand of heme 4 without significant alteration of the structure. A large increase of at least 200 mV of one of the four oxidation-reduction potentials was observed by electrochemistry and is interpreted in terms of structure/potential relationships.