Covalently bound flavin in the NqrB and NqrC subunits of Na⁺‐translocating NADH‐quinone reductase from Vibrio alginolyticus

Abstract

Na + -translocating NADH-quinone reductase (NQR) from the marine bacterium Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). On SDS^PAGE of the purified complex, NqrB and NqrC subunits were found to give yellowĝ reen fluorescent bands under UV illumination. Both the NqrB and NqrC, electroeluted from the gel, had an absorption maximum at 448 nm, and the fluorescence excitation maxima at 365 and 448 nm and the emission maximum at 514 nm. The electroeluted NqrB and NqrC, respectively, were identified from their N-terminal amino acid sequences. These results clearly indicated that the NqrB and NqrC subunits have covalently bound flavins. The two subunits were digested by protease and then the fluorescent peptide fragments were separated by a reversed-phase high performance liquid chromatography. NTerminal amino acid sequence analyses of the fluorescent peptides revealed that the flavin is linked to Thr-235 in the NqrB and Thr-223 in the NqrC subunits. This is the first example that the flavin is linked to a threonine residue. The amino acid sequence around the flavin-linked threonine was well conserved between NqrB and NqrC. Identification of the flavin group is in progress. z 2000 Federation of European Biochemical Societies.

Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration

“…A recent study of NosR from Paracoccus denitrificans shows agreement with the described topology (R. J. M. van Spanning, The flavin-binding domain of NosR is found in, among other proteins, the NqrC subunit of the Na ϩ -translocating NADH: quinone oxidoreductase of the genus Vibrio. NqrC was shown to carry FMN covalently bound as a phosphodiester to a threonine residue (17,27,52). Given the spectral evidence and the supporting flavin-binding consensus sequence, we suggest that NosR of P. stutzeri carries a flavin cofactor covalently bound to T163.…”

Electron Transport in the Pathway of Acetate Conversion to Methane in the Marine Archaeon Methanosarcina acetivorans

“…The MA0661 product shares identity with the RnfG subunit (Fig. 5C), which extends to a motif (ETPGLGX [37][38][39][40][41][42][43] GAT) that is also conserved with the NqrC subunit of Vibrio species (23) in which the C-terminal threonine of the motif covalently binds FMN (3,14,30). Therefore, the product of MA0661 is hypothesized to contain FMN, accepting electrons from the product of MA0659 (Fig.…”

A New Flavin Radical Signal in the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

“…The covalently bound FMNs in Na ϩ -NQR are bound to the protein by phosphoester bonds between the phosphate of the FMN and the -OH group of a threonine residue (Thr-236 in NqrB and Thr-225 in NqrC). In both cases the threonine is part of a highly conserved sequence of amino acids, SGAT (11,12,14). The role of these FMNs in electron transfer and in Na ϩ translocation has not been elucidated.…”