The nitrite reductase from Pseudomonas aeruginosa : Essential role of two active-site histidines in the catalytic and structural properties

Abstract

Cd1 nitrite reductase catalyzes the conversion of nitrite to NO in denitrifying bacteria. Reduction of the substrate occurs at the d1-heme site, which faces on the distal side some residues thought to be essential for substrate binding and catalysis. We report the results obtained by mutating to Ala the two invariant active site histidines, His-327 and His-369, of the enzyme from Pseudomonas aeruginosa. Both mutants have lost nitrite reductase activity but maintain the ability to reduce O 2 to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form, possibly because the productive displacement of NO from the ferric d 1-heme iron is impaired. Moreover, the two distal His play different roles in catalysis; His-369 is absolutely essential for the stability of the Michaelis complex. The structures of both mutants show (i) the new side chain in the active site, (ii) a loss of density of Tyr-10, which slipped away with the N-terminal arm, and (iii) a large topological change in the whole c-heme domain, which is displaced 20 Å from the position occupied in the wild-type enzyme. We conclude that the two invariant His play a crucial role in the activity and the structural organization of cd 1 nitrite reductase from P. aeruginosa.

Structure and Kinetic Properties of Paracoccus pantotrophus Cytochrome cd1 Nitrite Reductase with the d1 Heme Active Site Ligand Tyrosine 25 Replaced by Serine

“…Confirmation of this cyclization cannot be obtained from the crystal structure of the P. pantotrophus protein (wild-type or Y25S variant) because the first eight residues at the N terminus are disordered (Ref. 6 and see below). The 1.4-Å crystal structure of the Y25S variant (see below) clearly shows that there are no other amino acid changes throughout the protein, and the DNA sequence of the disordered part of the N-terminal arm region also indicates that it does not contain alterations in that region.…”

“…Ref. 6). Production of an inactive cd 1 in P. pantotrophus itself is problematic, as recent work on the expression of the nirS gene has shown that its transcription is activated by nitric oxide, the product of the reaction catalyzed by the enzyme (20).…”

NO Production by Pseudomonas aeruginosa cd1 Nitrite Reductase

“…Substitution of either of the two His with Ala has a dramatic effect on nitrite reduction, but only a marginal effect on the oxygen reductase activity (32).…”

Higher diversity and abundance of denitrifying microorganisms in environments than considered previously

“…The reduction of nitrite to NO is catalyzed by two structurally different but functionally equivalent nitrite reductases, that is, copper-containing reductase (NirK) and cytochrome cd1-containing reductase (NirS) (Hochstein and Tomlinson, 1988;Cutruzzola et al, 2001;Sakurai and Kataoka, 2007), and this is a key step in the denitrification process because dissolved N is converted into gaseous N during this step. Taxonomically diverse microorganisms have the ability to denitrify (Tiedje, 1994), and phylogenies based on the nir and 16S rRNA genes are incongruent (Jones et al, 2008).…”