Abstract
A key step in anaerobic nitrate respiration is the reduction of nitrite to nitric oxide, which is catalysed by the cd1 nitrite reductase NirS in, for example, the Gram-negative opportunistic pathogen Pseudomonas aeruginosa. Each subunit of this homodimeric enzyme consists of a cytochrome c domain and an eight-bladed β-propeller that binds the uncommon isobacteriochlorin heme d1 as an essential part of its active site. Although NirS has been well studied mechanistically and structurally, the focus of previous studies has been on the active heme d1-bound form. The heme d1-free form of NirS reported here, which represents a premature state of the reductase, adopts an open conformation with the cytochrome c domains moved away from each other with respect to the active enzyme. Further, the movement of a loop around Trp498 seems to be related to a widening of the propeller, allowing easier access to the heme d1-binding side. Finally, a possible link between the open conformation of NirS and flagella formation in P. aeruginosa is discussed.
Highlights
Denitrification is the stepwise reduction of nitrogen oxides to dinitrogen and is utilized by many bacteria to replace the terminal oxygen-dependent steps of the respiratory chain under anaerobic conditions
In previous work with the dihydro-heme d1 dehydrogenase NirN, we isolated the cd1 nitrite reductase NirS with bound dihydro-heme d1, the final intermediate of heme d1 biosynthesis, which differs from heme d1 by lacking a double bond in the propionate chain attached to ring D (Klunemann et al, 2019)
Comparison with the previously published heme d1-bound structure of NirS reveals no significant differences (Nurizzo et al, 1997; C r.m.s.d. of 0.38 A ; Figs. 3 and 4). This suggests that the reported differences in the activity of NirS loaded with dihydro-heme d1 or heme d1 are likely to be caused by inherent properties of the cofactor
Summary
Denitrification is the stepwise reduction of nitrogen oxides to dinitrogen and is utilized by many bacteria to replace the terminal oxygen-dependent steps of the respiratory chain under anaerobic conditions. Many bacteria utilize the cd nitrite reductase NirS to catalyse this step, and the NirS enzymes from Pseudomonas aeruginosa (Pa-NirS) and Paracoccus pantotrophus (Pp-NirS) have been well studied functionally and structurally Both chains of this homodimeric enzyme possess a c-type cytochrome domain with a covalently attached heme c functioning as an electron-entry point and a C-terminal eight-bladed -propeller that utilizes the uncommon isobacteriochlorin heme d1 as an active-site cofactor (Fig. 1a; Nurizzo et al, 1997; Williams et al, 1997). Compared with other tetrapyrroles such as heme b, a higher affinity for anionic molecules such as nitrite and a lower affinity for nitric oxide has been observed for the ferrous isobacteriochlorin heme d1 This is rooted in the unique carbonyl moieties at rings A and B and the replacement of the typical propionate by an acrylate at ring D (Chang et al, 1986; Rinaldo et al, 2011; Fujii et al, 2016).
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