The TT1485 gene from Thermus thermophilus HB8 encodes a hypothetical protein of unknown function with about 20 sequence homologs of bacterial or archaeal origin. Together they form a family of uncharacterized proteins, the cluster of orthologous group COG3253. Using a combination of amino acid sequence analysis, three-dimensional structural studies and biochemical assays, we identified TT1485 as a novel heme-binding protein. The crystal structure reveals that this protein is a pentamer and each monomer exhibits a beta-barrel fold. TT1485 is structurally similar to muconolactone isomerase, but this provided no functional clues. Amino acid sequence analysis revealed remote homology to a heme enzyme, chlorite dismutase. Strikingly, amino acid residues that are highly conserved in the homologous hypothetical proteins and chlorite dismutase cluster around a deep cavity on the surface of each monomer. Molecular modeling shows that the cavity can accommodate a heme group with a strictly conserved His as a heme ligand. TT1485 reconstituted with iron protoporphyrin IX chloride gave a low chlorite dismutase activity, indicating that TT1485 catalyzes a reaction other than chlorite degradation. The presence of a possible Fe-His-Asp triad in the heme proximal site suggests that TT1485 functions as a novel heme peroxidase to detoxify hydrogen peroxide within the cell.
Heme-thiolate protein model complexes, [FeIII(OEP)(Ac-LcXAF-LLLLL-ALFL-OMe)] {OEP = octhaethylporphinato, X = Leu (1) and Pro (2)}, having α-helical structure were synthesized and characterized as P-450 and CPO models. The stable ligation of cysteinyl thiolate at the axial position of model complexes was established using MCD, UV−visible, 1H NMR, and ESI-MS. These results indicate that 1 and 2 possess high-spin 5-coordinated Fe(III) ions. The helical contents were examined by CD measurements in THF which revealed the contents of 1 and [GaIII(OEP)(Ac-LcLAF-LLLLL-ALFL-OMe)] (3) to be both 49%, though the thiolate anion (Et4N){Ac-LC(S-)LAF-LLLLL-ALFL-OMe} (5) has the value of 57%. Thus, the α-helical NH···OC hydrogen bond network is intercepted by the NH···S hydrogen bond between NH(Leu3) and Sγ(Cys2). However, the helical percentages of 2 (59%) and [GaIII(OEP)(Ac-LcPAF-LLLLL-ALFL-OMe)] (4) (57%) are similar to that of (Et4N){Ac-LC(S-)LAF-LLLLL-ALFL-OMe} (6) (52%). The NMR analysis of the solution structure of the diamagnetic Ga(III) complex 4 indicates the distances between CysS γ and NH of the third residue to be 2.37 Å, which is shorter than those of [GaIII(OEP)(Z-Cys-Pro-Ala-Leu-OMe)] (2.58 Å) and [GaIII(OEP)(Z-Cys-Pro-Leu-OMe)] (3.00 Å). These results show that the NH···S hydrogen bond of 4 is shortened by α-helix dipole and then the α-helix is stabilized. The redox potentials of FeIII/FeII for 1 and 2 are −0.54 and −0.55 V (vs SCE in CH2Cl2), respectively. The values are more positively shifted from those of [FeIII(OEP)(Z-Cys-Leu-Gly-OMe)] (−0.61 V vs SCE) and [FeIII(OEP)(Z-Cys-Pro-Leu-OMe)] (−0.68 V vs SCE). Complex 1 indicates the positive shift of only 70 mV because the Cys-Leu-Ala fragment on the N terminus breaks the α-helical conformation. In contrast, complex 2 has larger positive shift (130 mV) by the α-helical conformation cooperating with the NH···S hydrogen bond. These data suggest that the redox reactions are regulated by the cooperating effect of the α-helix and the NH···S hydrogen bonds in the native enzymes.
Recombinant holo Thermus thermophilus [7Fe-8S] ferredoxin was synthesized by cloning from Thermus thermophilus HB8 gene. A specific sequence (Pro-His-Val-Ile) at the N-terminus of the recombinant ferredoxin was determined by a rapid and highly sensitive mass spectral method using a novel Ru(II) Edman reagent, [(tpy)Ru(tpy-C6H4-NCS)](PF6)2 (tpy=terpyridine). The formation of the recombinant holoTtFd was established by the characteristic absorptions and CD extrema as [7Fe-8S] ferredoxin. The catalytic electron-transfer reactivity of the [7Fe-8S] ferredoxin between ferredoxin-NADP+ reductase and cytochrome c was recognized.
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