The hydrogenase cytochrome b heme ligands of Azotobacter vinelandii are required for full H-2 oxidation capability

Authors
Citation
L. Meek et Dj. Arp, The hydrogenase cytochrome b heme ligands of Azotobacter vinelandii are required for full H-2 oxidation capability, J BACT, 182(12), 2000, pp. 3429-3436
Citations number
50
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Microbiology
Journal title
JOURNAL OF BACTERIOLOGY
ISSN journal
0021-9193 → ACNP
Volume
182
Issue
12
Year of publication
2000
Pages
3429 - 3436
Database
ISI
SICI code
0021-9193(200006)182:12<3429:THCBHL>2.0.ZU;2-8
Abstract
The hydrogenase in Azotobacter vinelandii, like other membrane-bound [NiFe] hydrogenases, consists of a catalytic heterodimer and an integral membrane cytochrome b. The histidines ligating the hemes in this cytochrome b were identified by H-2 oxidation properties of altered proteins produced by site -directed mutagenesis. Four fully conserved and four partially conserved hi stidines in HoxZ were substituted with alanine or tyrosine, The roles of th ese histidines in HoxZ heme binding and hydrogenase were characterized by O -2-dependent H-2 oxidation and H-2-dependent methylene blue reduction in vi vo. Mutants H33A/Y (H33 replaced by A or Y), H74A/Y, H194A, H208A/Y, and H1 94,208A lost O-2-dependent H-2 oxidation activity, H194Y and H136A had part ial activity, and H97Y,H98A and H191A had full activity. These results sugg est that the fully conserved histidines 33, 74, 194, and 208 are ligands to the hemes, tyrosine can serve as an alternate ligand in position 194, and H136 plays a role in H-2 oxidation, In mutant H194A/Y, imidazole (Imd) resc ued H-2 oxidation activity in intact cells, which suggests that Imd acts as an exogenous ligand. The heterodimer activity, quantitatively determined a s H-2-dependent methylene blue reduction, indicated that the heterodimers o f all mutants were catalytically active. H33A/Y had wild-type levels of met hylene blue reduction, but the other HoxZ ligand mutants had significantly less than wild-type levels. Imd reconstituted full methylene blue reduction activity in mutants H194A/Y and H208A/Y and partial activity in H194,208A. These results indicate that structural and functional integrity of HoxZ is required for physiologically relevant H-2 oxidation, and structural integr ity of HoxZ is necessary for full heterodimer-catalyzed H-2 oxidation.