Identification of the Ferredoxin-Binding Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase

Abstract

An in silico model for the 1:1 ferredoxin (Fd)/nitrate reductase (NR) complex, using the known structure of Synechocystis sp. PCC 6803 Fd and the in silico model of Synechococcus sp. PCC 7942 NR, is used to map the interaction sites that define the interface between Fd and NR. To test the electrostatic interactions predicted by the model complex, five positively charged NR amino acids (Arg43, Arg46, Arg197, Lys201, and Lys614) and a negatively charged amino acid (Glu219) were altered using site-directed mutagenesis and characterized by activity measurements, metal analysis, and electron paramagnetic resonance (EPR) studies. All of the charge replacement variants retained wild-type levels of activity with reduced methyl viologen (MV), but a significant decrease in activity was observed for the R43Q, R46Q, K201Q, and K614Q variants when reduced Fd served as the electron donor. EPR analysis as well as the Fe and Mo analyses showed that loss of activity observed with these variants was not the consequence of perturbation of the Mo center or [4Fe-4S] cluster. Therefore, the loss of the Fd-linked specific activity observed with these variants can be explained only by invoking a role for Arg43, Arg46, Lys201, and Lys614 in Fd binding. The R43Q, R46Q, K201Q, and K614Q NR variants also showed a decreased binding affinity for Fd, compared to that of wild-type NR, supporting a key role of these four positively charged residues in the productive binding of Fd.

Structural basis for electron transport mechanism of complex I-like photosynthetic NAD(P)H dehydrogenase

“…4a-c), thus is essential for Fd binding. This structural feature is consistent with previous studies showing that Fd features a highly acidic surface, with recruitment usually driven by basic patches on the binding partner [46][47][48] . Furthermore, our surface plasmon resonance (SPR) results demonstrated that the binding affinity of NDH-1L with Fd is significantly increased at higher pH ( Supplementary Fig.…”

A recently evolved diflavin-containing monomeric nitrate reductase is responsible for highly efficient bacterial nitrate assimilation

“…The holoenzyme of the canonical NAS enzymes is hetero-oligomeric in nature, composed of one conserved catalytic subunit and at least one electron transfer subunit (which may be shared with the nitrite reductase) or one electron donor subunit (ferredoxin or flavodoxin) (Fig. 1), wherein the [2Fe-2S] cluster plays a crucial role, universally, in intermolecular electron transfer for nitrate reduction (8,13). In contrast, although the N-terminal region of NasN contains a canonical CA domain, it is NADPH-dependent and lacks the [2Fe-2S] cluster-binding domain.…”

“…1), represented by the well-studied enzyme from the terrestrial diazotroph Azotobacter vinelandii (6), directly acquires electrons from flavodoxin via its C-terminal [2Fe-2S] cluster. In contrast, the ferredoxin-dependent heterodimeric NarB (Fd-NarB) acquires electrons from the photosynthetically reduced ferredoxin containing a [2Fe-2S] cluster, as identified in the autotrophic cyanobacterium Synechococcus elongatus (7,8). Thus, the electron donor (flavodoxin or ferredoxin) functions as a subunit of NarB holoenzymes.…”

“…Others are heterotrimeric complexes, well-represented by Paracoccus denitrificans NasBGC, composed of a NasA-like catalytic subunit, a FAD/NAD-containing nitrite reductase subunit, and a [2Fe-2S] cluster-containing electron transfer subunit (13). It is worth noting that the [2Fe-2S] cluster plays an essential role in intermolecular electron transfer for nitrate reduction catalyzed by all of these canonical NASs (8,13). In addition, both heterodimeric and heterotrimeric NADH-dependent NASs carry a FAD/NAD domain with NADH-and NADPH-binding sites.…”