The underlying stereochemical mechanisms for the dramatic differences in autooxidation and hemin loss rates of fish versus mammalian hemoglobins (Hb) have been examined by determining the crystal structures of perch, trout IV, and bovine Hb at high and low pH. The fish Hbs autooxidize and release hemin ∼50 to 100-fold more rapidly than bovine Hb. Five specific amino acid replacements in the CD corner and along the E helix appear to cause the increased susceptibility of fish Hbs to oxidative degradation compared to mammalian Hbs. Ile is present at the E11 helical position in most fish Hb chains whereas a smaller Val residue is present in all mammalian α and β chains. The larger IleE11 side chain sterically hinders bound O 2 and facilitates dissociation of the neutral superoxide radical, enhancing autooxidation. Lys(E10) is found in most mammalian Hb and forms favorable electrostatic and hydrogen bonding interactions with the heme-7-propionate. In contrast, Thr(E10) is present in most fish Hbs and is too short to stabilize bound heme, and causes increased rates of hemin dissociation. The especially high rates of hemin loss in perch Hb are also due to a lack of electrostatic interaction between His(CE3) and the heme-6 propionate in α subunits whereas this interaction does occur in trout IV and bovine Hb. There is also a larger gap for solvent entry into the heme crevice near β CD3 in the perch Hb (∼8Å) compared to trout IV Hb (∼6Å) which in turn is significantly higher than that in bovine Hb (∼4Å) at low pH. The amino acids at CD4 and E14 differ between bovine and the fish Hbs and have the potential to modulate oxidative degradation by altering the orientation of the distal histidine and the stability of the E-helix. The generally rapid rates of lipid oxidation in fish muscle can be partly attributed to the fact that fish Hbs are highly susceptible to oxidative degradation.
The results of this study aid in the selection of an appropriate DNA extraction kit for a given soil sample. Its application could expedite sample processing for real-time PCR detection of a pathogen in soil.
Surface proteins Shr, Shp, and the ATP-binding cassette (ABC) transporter HtsABC are believed to make up the machinery for heme uptake in Streptococcus pyogenes. Shp transfers its heme to HtsA, the lipoprotein component of HtsABC, providing the only experimentally demonstrated example of direct heme transfer from a surface protein to an ABC transporter in Gram-positive bacteria. To understand the structural basis of heme transfer in this system, the heme-binding domain of Shp (Shp(180)) was crystallized, and its structure determined to a resolution of 2.1 A. Shp(180) exhibits an immunoglobulin-like beta-sandwich fold that has been recently found in other pathogenic bacterial cell surface heme-binding proteins, suggesting that the mechanisms of heme acquisition are conserved. Shp shows minimal amino acid sequence identity to these heme-binding proteins and the structure of Shp(180) reveals a unique heme-iron coordination with the axial ligands being two methionine residues from the same Shp molecule. A negative electrostatic surface of protein structure surrounding the heme pocket may serve as a docking interface for heme transfer from the more basic outer cell wall heme receptor protein Shr. The crystal structure of Shp(180) reveals two exogenous, weakly bound hemins, which form a large interface between the two Shp(180) molecules in the asymmetric unit. These "extra" hemins form a stacked pair with a structure similar to that observed previously for free hemin dimers in aqueous solution. The propionates of the protein-bound heme coordinate to the iron atoms of the exogenous hemin dimer, contributing to the stability of the protein interface. Gel filtration and analytical ultracentrifugation studies indicate that both full-length Shp and Shp(180) are monomeric in dilute aqueous solution.
The surface protein Shp of Streptococcus pyogenes rapidly transfers its hemin to HtsA, the lipoprotein component of the HtsABC transporter, in a concerted two-step process with one kinetic phase. The structural basis and molecular mechanism of this hemin transfer have been explored by mutagenesis and truncation of Shp. The heme-binding domain of Shp is in the amino-terminal region and is functionally active by itself, although inclusion of the COOH-terminal domain speeds up the process ϳ10-fold. . Thus, the M66A and M153A replacements alter the kinetic mechanism and unexpectedly slow down hemin transfer by stabilizing the intermediates. These results, in combination with the structure of the Shp heme-binding domain, allow us to propose a "plug-in" mechanism in which side chains from apoHtsA are inserted into the axial positions of hemin in Shp to extract it from the surface protein and pull it into the transporter active site.Numerous acquisition machineries have been identified in bacterial pathogens for heme as a preferred iron source from mammals. Specific ATP-binding cassette (ABC) 2 transporters, which transport heme across the cytoplasmic membrane, are common components of the uptake machineries in both Grampositive and Gram-negative pathogens (1-3). However, the transfer events and proteins involved prior to the action of the ABC transporters are different due to the distinct extracellular structures between these two types of bacteria. Gram-negative bacteria utilize an outer membrane receptor protein to acquire heme from host hemoproteins directly or through a hemophore and bring the captured heme to the periplasmic space for the ABC transporter in a TonB-dependent process (4 -6). Gram-positive bacteria produce cell surface proteins to relay heme from host proteins to the ABC transporter (7-9).The Fe(II)-protoporphyrin IX complex (heme) or Fe(III)-protoporphyrin IX complex (hemin) exchange from one protein to another has been demonstrated biochemically in only a few bacterial systems, including transfers from hemoglobin to Serratia marcescens hemophore HasA (10); from the cell surface protein Shp to HtsA, the lipoprotein component of the HtsABC transporter, in Streptococcus pyogenes and Streptococcus equi (11,12); from HasA to its outer membrane receptor HasR (10); and from hemoglobin to Shigella dysenteriae outer membrane receptor ShuA (5). A detailed kinetic mechanism has only been proposed for the S. pyogenes Shp/HtsA system (13). This process occurs in a single kinetic phase with transfer rate constants that are ϳ100,000 times greater than that for simple hemin dissociation from Shp. The structural basis for this rapid and concerted heme transfer is unknown.In some hemoproteins, iron is hexacoordinate, with four ligands from protoporphyrin IX and two axial ligands from the side chains of His, Lys, Tyr, Met, and/or Cys. Combinations of the strong ligands, His, Lys, Met, and Cys, usually result in the * This work was supported by National Institutes of Health Grants AI057347
Picosecond time-resolved crystallography was used to follow the dissociation of carbon monoxide from the heme pocket of a mutant sperm whale myoglobin and the resultant conformational changes. Electron-density maps have previously been created at various time points and used to describe amino-acid side-chain and carbon monoxide movements. In this work, difference refinement was employed to generate atomic coordinates at each time point in order to create a more explicit quantitative representation of the photo-dissociation process. After photolysis the carbon monoxide moves to a docking site, causing rearrangements in the heme-pocket residues, the coordinate changes of which can be plotted as a function of time. These include rotations of the heme-pocket phenylalanine concomitant with movement of the distal histidine toward the solvent, potentially allowing carbon monoxide movement in and out of the protein and proximal displacement of the heme iron. The degree of relaxation toward the intermediate and deoxy states was probed by analysis of the coordinate movements in the time-resolved models, revealing a non-linear progression toward the unbound state with coordinate movements that begin in the heme-pocket area and then propagate throughout the rest of the protein.
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