Heme Axial Ligation by the Highly Conserved His Residues in Helix II of Cytochrome b (NarI) of Escherichia coliNitrate Reductase A (NarGHI)

Abstract

Nitrate reductase A (NarGHI) 1 is the predominant respiratory complex in the membrane of Escherichia coli when the cells are grown under anaerobic conditions in the presence of nitrate. This complex catalyzes electron transfer from quinol to nitrate coupled to proton release into the periplasm and the generation of a concomitant transmembrane proton electrochemical potential (1). NarGHI is a complex of three nonidentical subunits, NarG (150 kDa), NarH (60 kDa), and NarI (25 kDa), in the ratio of 1:1:1 as predicted by the operon organization. This complex is arranged in two catalytic domains: the NarGH subunits constitute the cytoplasmic domain and NarI the membrane anchor domain (2, 3). The NarGH domain contains four [Fe-S] centers (4, 5) and a molybdenum cofactor which is the site of nitrate reduction by the enzyme (6). The NarI subunit (cytochrome b nr ) is a diheme b-type cytochrome required for attachment of the cytoplasmic domain to the inner side of the cytoplasmic membrane (3) and it mediates electron transfer from the membrane quinol pool (menaquinol or ubiquinol) to the molybdenum cofactor (2, 7). Optical redox titrations demonstrate the presence of two hemes in NarGHI with midpoint potentials (E m,7 values) of ϩ17 and ϩ122 mV (8).Of the well characterized membrane-intrinsic cytochromes b, the hemes are found to have bis-histidine ligation (9 -11), and it is very likely that this is also the case with the hemes of NarGHI. The hydropathy plot 2 and sequence analysis of NarI using the "positive inside" rule of von Heijne (12) leads to a topological model in which five transmembrane ␣-helical segments (helices I-V) are arranged with a periplasmic N terminus and a cytoplasmic C terminus (13). Five His residues are present in predicted transmembrane segments, at positions 56, 66, 74, 187, and 205. Sequence alignment of all known sequences of NarI from various bacterial membrane-bound nitrate reductases (4, 13, 14) reveals only four totally conserved histidines (His-56,His-66 in helix II and His-187,His-205 in helix V) as potential heme ligands, thereby excluding His-74 within helix II in heme ligation (13). Overall, this strongly suggests ligation of each heme by two histidine residues (13). Because of the different spacing of the conserved histidines on helix II (9 residues) and V (17 residues), the hemes probably lie in two planes with different angles relative to the plane of the membrane. Recently, an alternative model for heme ligation in NarI has been proposed by van der Oost et al. (15) on the basis of subunit stoichiometry and sequence analysis of various b-type cytochromes. This model predicts that the hemes are sandwiched in a NarI dimer and ligated only by His-187 and His-205 of helix V of each NarI subunit. Genetic procedures are, therefore, required for identifying the heme ligands and to distinguish the different models.In the present work, we have used site-directed mutagenesis, as well as optical and EPR spectroscopies, to test whether the two conserved histidine residues within helix II (Hi...

The Molybdenum Cofactor of Escherichia coli Nitrate Reductase A (NarGHI)

“…In contrast to the conclusions drawn from earlier studies, our results with a mobAB mutant indicate that nucleotide attachment and metal chelation must precede cofactor insertion into NarGH. Since both the soluble (NarGH) (20,22) and membrane-bound (NarGHI) (24,40) forms of the enzyme can readily be studied and appear to contain Mo-MGD in a wild-type background, cofactor insertion most likely occurs prior to attachment of the NarGH dimer to the membrane anchor subunit (NarI). In the presence of tungstate or in a mobAB mutant background, it is clear that attachment of cofactor-deficient NarGH to the membrane-bound NarI The accumulation molybdate appears to be dependent on the mod operon (37,38) and the poorly characterized mog locus (42) that may encode a molybdochelatase (43).…”

Axial Ligation and Stoichiometry of Heme Centers in Adrenal Cytochromeb561

“…Similar low-spin signals are known from studies of other cytochromes. An EPR signal with g z = 2.96 was noted as a minor species in some samples of cyt b 561 (20,21,43,60) and in unrelated systems with a mutated cytochrome b component (61,62). This signal has been identified as "relaxed" conformation of b-type cytochrome in which the strain on heme ligation imposed by the native protein is removed (63).…”

Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617

“…The midpoint redox potentials of the hemes were E m7.6 = ?197 mV (heme c) and E m7.6 = -4.5 mV (heme b). In E. coli and P. denitrificans enzymes, two different b-type hemes (b L and b H ) were identified [11,46,47] and Fig. 1 Electrophoretic analysis of NarGH and NarGHI enzyme complexes isolated from Marinobacter hydrocarbonoclasticus 617: A and G low molecular weight markers (Amersham Biosciences); B NarGH, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE; 10%), stained with Coomassie Blue R-250: C NarGH, native PAGE, stained with Coomassie blue R-250; D NarGH, native PAGE, stained for respiratory nitrate reductase (Nar) activity; E NarGHI, native PAGE; stained for Nar activity; F NarGHI, SDS-PAGE (12.5%), stained with Coomassie blue R-250 their midpoint redox potentials are presented in Table 1 for comparison.…”