Redox cofactors insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism

Abstract

A major gap of knowledge in metalloproteins is the identity of the prefolded state of the protein before cofactor insertion. This holds for molybdoenzymes serving multiple purposes for life, especially in energy harvesting. This large group of prokaryotic enzymes allows for coordination of molybdenum or tungsten cofactors (Mo/W-bisPGD) and Fe/S clusters. Here we report the structural data on a cofactor-less enzyme, the nitrate reductase respiratory complex and characterize the conformational changes accompanying Mo/W-bisPGD and Fe/S cofactors insertion. Identified conformational changes are shown to be essential for recognition of the dedicated chaperone involved in cofactors insertion. A solvent-exposed salt bridge is shown to play a key role in enzyme folding after cofactors insertion. Furthermore, this salt bridge is shown to be strictly conserved within this prokaryotic molybdoenzyme family as deduced from a phylogenetic analysis issued from 3D structure-guided multiple sequence alignment. A biochemical analysis with a distantly-related member of the family, respiratory complex I, confirmed the critical importance of the salt bridge for folding. Overall, our results point to a conserved cofactors insertion mechanism within the Mo/W-bisPGD family.

A dual functional redox enzyme maturation protein for respiratory and assimilatory nitrate reductases in bacteria

“…Firstly, a sequence 11 CGVGCGV 17 is present within the N‐terminal/proximal iron‐sulfur cluster binding motif in P. denitrificans NasC and other assimilatory reductases that has similarity to the 49 H/CGVNCTG 55 sequence required for FS0 insertion and subsequent Mo[PGD] 2 loading for E. coli NarG. (Lanciano et al , ; Arias‐Cartin et al , ) Secondly, further residues that may support formation of an arginine‐glutamate salt‐bridge identified by Arias‐Cartin and co‐workers for correct protein‐folding of Mo/W‐[PGD] 2 family holoenzymes, (Arias‐Cartin et al , ) are also conserved in NasC (i.e. R58 and E437).…”

“…N‐terminal [4Fe‐4S] cluster coordinating residues are highlighted in black. Regions shaded in blue and red show predicted sequence regions required for [4Fe‐4S] (and subsequent Mo[PGD] 2 ) loading and a conserved solvent‐exposed salt bridge in Mo/W [PGD] 2 family proteins (Arias‐Cartin et al , ), respectively. Asterisks denote the conserved R58 and E437 residues for P. denitrificans NasC.…”

Perturbation of formate pathway and NADH pathway acting on the biohydrogen production

“…Therefore, many genetic engineering studies about the FHL-related genes have been done to regulate the metabolism of the formate pathway to enhance the hydrogen production (Supplementary Table S2 ). These operations include inactivation of hycA (a negative regulator) 25 and focA (formate transmembrane protein) 26 , as well as overexpression of fhlA (a transcriptional activator) 27 , 28 , fdhF (formate dehydrogenase H) 29 , modE (a transcriptional activator) 26 and selC (tRNA related to formate dehydrogenase) 26 .…”

“…In anaerobic metabolism, NADH generated from glycolysis is mainly consumed by the lactate production pathway and the alcohol production pathway, so the disruption of the lactate dehydrogenase gene ldhA and the alcohol dehydrogenase gene adh to increase H 2 production is feasible 30 . Some other genes or gene clusters related to the NADH metabolism such as frdBC 16 , nuoB 29 , nadE 15 and hoxEFUYH 31 have also been regulated to improve the hydrogen yields.…”

History of Maturation of Prokaryotic Molybdoenzymes—A Personal View

“…Indeed, while the lack of Moco does not preclude FS0 insertion, the absence of NarJ or substitution of cysteine ligands of the FS0 cluster prevents the insertion both of FS0 and of Moco in NarG [61,63]. In our search for Moco-driven conformational changes, we were successful in isolating and characterizing a stable prefolded state that remains competent for cofactor insertion, like that of the cytoplasmic and soluble apoNarGH complex produced in the absence of NarJ [71]. It is worth recalling that this assembly intermediate contains all four FeS clusters in the NarH subunit.…”