Reduction of the off-pathway iron-sulphur cluster N1a of Escherichia coli respiratory complex I restrains NAD+ dissociation

Abstract

Respiratory complex I couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. The reaction starts with NADH oxidation by a flavin cofactor followed by transferring the electrons through a chain of seven iron-sulphur clusters to quinone. An eighth cluster called N1a is located proximally to flavin, but on the opposite side of the chain of clusters. N1a is strictly conserved although not involved in the direct electron transfer to quinone. Here, we show that the NADH:ferricyanide oxidoreductase activity of E. coli complex I is strongly diminished when the reaction is initiated by an addition of ferricyanide instead of NADH. This effect is significantly less pronounced in a variant containing N1a with a 100 mV more negative redox potential. Detailed kinetic analysis revealed that the reduced activity is due to a lower dissociation constant of bound NAD+. Thus, reduction of N1a induces local structural rearrangements of the protein that stabilise binding of NAD+. The variant features a considerably enhanced production of reactive oxygen species indicating that bound NAD+ represses this process.

Cryo-EM structures reveal intricate Fe-S cluster arrangement and charging in Rhodobacter capsulatus formate dehydrogenase

“…2b). This cluster was suggested to minimise reactive oxygen species (ROS) production due to flavin semiquinone radicals in the presence of oxygen by temporarily storing electrons 26 , inducing structural rearrangements that stabilise NAD + binding upon reduction 28 , or increasing overall enzyme stability 29 . Although the diaphorase structure of RcFDH shares high similarity with those of other oxidoreductases, the amino-terminal ferredoxin-like domain of FdsB is absent in related NADH-quinone oxidoreductases and the NAD + -reducing [NiFe] hydrogenases ( Supplementary Fig.…”

A mechanism to prevent production of reactive oxygen species by Escherichia coli respiratory complex I

“…However, the ferricyanide-initiated NADH/ferricyanide oxidoreductase activity of E. coli complex I is less than 10% of the rate obtained by starting the reaction with NADH 30–32 . This was interpreted as a local structural rearrangement at the NADH-binding site 30,31 or as dissociation of FMN from the reduced enzyme 32 . The latter proposal is highly unlikely as the FMN cofactor is bound in both, the oxidized and reduced state of NuoEF, with full occupancy, low B-factors and experiences no change in its binding (Figs 2, 3).…”

“…6). The acceleration of the reaction is interpreted as a slightly faster oxidation of N1a during NADH/ferricyanide oxidoreductase activity than its subsequent reduction by NADH 31 . A detailed kinetic analysis revealed that the slow rate of the ferricyanide-initiated reaction is due to an enhanced binding of the reaction product, NAD + 31 .…”

Iron‐sulfur cluster carrier proteins involved in the assembly of Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

“…An additional binuclear cluster on NuoE, N1a, is not part of the chain of clusters but is located in electron transfer distance to FMN. The cluster is strictly conserved and plays not only a role in the stability and the assembly of the complex (Birrell et al, 2013;Dörner et al, 2017) but also in regulating NADH oxidation (Gnandt et al, 2017).…”