Site-specific mutational analysis of a novel cysteine motif proposed to ligate the 4Fe-4S cluster in the iron-sulfur flavoprotein of the thermophilicmethanoarchaeon Methanosarcina thermophila

Isf (iron-sulfur flavoprotein) from Methanosarcina thermophila has been pro duced in Escherichia coli as a dimer containing two 4Fe-4S clusters and two FMN (flavin mononucleotide) cofactors, The deduced sequence of Isf contain s six cysteines (Cys 16, Cys 47, Cys 50, Cys 53, Cys 59, and Cys 180), four of which (Cys 47, Cys 50, Cys 53, and Cys 59) comprise a motif with high i dentity to a motif (CX2CX2CX4-7C) present in all homologous Isf sequences a vailable in the databases. The spacing of the motif is highly compact and a typical of motifs coordinating known 4Fe-4S clusters; therefore, all six cy steines in Isf from M. thermophila were altered to either alanine or serine to obtain corroborating biochemical evidence that the motif coordinates th e 4Fe-4S cluster and to further characterize properties of the cluster depe ndent on ligation, All except the C16S variant were produced in inclusion b odies and were void of iron-sulfur clusters and FMN, Reconstitution of the iron-sulfur cluster and FMN was attempted for each variant. The UV-visible spectra of all reconstituted variants indicated the presence of iron-sulfur clusters and FMN. The reduced C16A/S variants showed the same electron par amagnetic resonance (EPR) spectra as wild-type Isf, whereas the reduced C18 0A/S variants showed EPR spectra identical to those of one of the two 4Fe-4 S species present in the wild-type Isf spectrum. Conversely, EPR spectra of the oxidized C50A and C59A variants showed g values characteristic of a 3F e-4S cluster. The spectra of the C47A and C53A variants indicated a 4Fe-4S cluster with g values and linewidths different from those for the mild type . The combined results of this study support a role for the novel CX2CX2CX4 -7C motif in ligating the 4Fe-4S clusters in Isf and Isf homologues.