Histidine 131, not histidine 43, of the Bradyrhizobium japonicum FixN protein is exposed towards the periplasm and essential for the function of the cbb₃‐type cytochrome oxidase

Abstract

In subunit I (FixN) of the Bradyrhizobiumjaponicum cbb3-type oxidase, only five instead of the normal six strictly conserved histidines (H) could be unambiguously assigned as the putative heme or copper ligands. The ambiguity concerned H43 or H131 as the presumptive N-terminal ligands of the low-spin heme B. We report here that a H43A replacement had a wildtype phenotype, whereas the H131A mutant was defective in oxidase function and suhunlt assembly or stability, suggesting that H131 serves as the N-terminal low-spin heine ligand. Topological studies revealed that H131 resides on the periplasmic side of helix 2, where one of the low-spin heme ligands is normally found in conventional heme-copper oxidases.

Isolation and Characterization of Rhodobacter capsulatus Mutants Affected in Cytochrome cbb 3 Oxidase Activity

“…In addition, our current data in combination with previous work on M7G (15) suggest that the low-spin heme group is also not essential for the stability of R. capsulatus cyt cbb 3 oxidase, as it has been shown for R. sphaeroides cyt aa 3 oxidase (19). Again, this is unlike B. japonicum, where the cyt cbb 3 oxidase becomes unstable if its putative low-spin heme ligand H131 is substituted for by an alanine (58). Taken together, the data presented in this work clearly suggest an important role for CcoP in the assembly and activity of the cyt cbb 3 oxidase in R. capsulatus, unlike in B. japonicum, where this subunit appears to be dispensable.…”

A Cytochrome cbb 3 (Cytochrome c) Terminal Oxidase in Azospirillum brasilense Sp7 Supports Microaerobic Growth

“…The highly conserved histidine residues shown to be involved in the binding of the high-spin b/Cu B reaction center (5,26) are conserved at positions 362, 274, 275, and 224 in the A. brasilense CytN. The histidine residues assumed to be the axial ligands for the low-spin heme b (22,56) 56hypothesized that the first 2 of these 14 transmembrane helices of CytN-like proteins should be cytoplasmic. This hypothesis was supported by studies with fusion proteins (56).…”

“…The histidine residues assumed to be the axial ligands for the low-spin heme b (22,56) 56hypothesized that the first 2 of these 14 transmembrane helices of CytN-like proteins should be cytoplasmic. This hypothesis was supported by studies with fusion proteins (56). Interestingly the A. brasilense CytN protein seems to be truncated and lacks these two first transmembrane helices encountered in other sequenced CytN-like proteins.…”

How Replacements of the 12 Conserved Histidines of Subunit I Affect Assembly, Cofactor Binding, and Enzymatic Activity of the Bradyrhizobium japonicum cbb 3-type Oxidase

“…Subunit I (FixN in B. japonicum), the largest subunit of the cbb 3 -type oxidase, is a 12-transmembrane-helix protein with a molecular mass of 61 kDa and contains two hemes of the B type and a Cu B (1,14,15). The high spin heme B forms a binuclear center with the Cu B metal (15,16).…”