Iron-Sulfur Flavoprotein (Isf) from Methanosarcina thermophila Is the Prototype of a Widely Distributed Family

Abstract

A total of 35 homologs of the iron-sulfur flavoprotein (Isf) from Methanosarcina thermophila were identified in databases. All three domains were represented, and multiple homologs were present in several species. An unusually compact cysteine motif ligating the 4Fe-4S cluster in Isf is conserved in all of the homologs except two, in which either an aspartate or a histidine has replaced the second cysteine in the motif. A phylogenetic analysis of Isf homologs identified four subgroups, two of which were supported by bootstrap data. Three homologs from metabolically and phylogenetically diverse species in the Bacteria and Archaea domains (Af3 from Archaeoglobus fulgidus, Cd1 from Clostridium difficile, and Mj2 from Methanococcus jannaschii) were overproduced in Escherichia coli. Each homolog purified as a homodimer, and the UV-visible absorption spectra were nearly identical to that of Isf. After reconstitution with iron, sulfide, and flavin mononucleotide (FMN) the homologs contained six to eight nonheme iron atoms and 1.6 to 1.7 FMN molecules per dimer, suggesting that two 4Fe-4S or 3Fe-4S clusters and two FMN cofactors were bound to each dimer, which is consistent with Isf data. Homologs Af3 and Mj2 were reduced by CO in reactions catalyzed by cell extract of acetate-grown M. thermophila, but Cd1 was not. Homologs Af3 and Mj2 were reduced by CO in reactions catalyzed by A. fulgidus and M. jannaschii cell extracts. Cell extract of Clostridium thermoaceticum catalyzed CO reduction of Cd1. Our database sequence analyses and biochemical characterizations indicate that Isf is the prototype of a family of iron-sulfur flavoproteins that occur in members of all three domains.Methane produced by the acetate fermentation pathway accounts for two-thirds of all biologically produced methane. In Methanosarcina thermophila, acetate is cleaved into carbonyl and methyl groups by the CO dehydrogenase (CODH)-acetyl coenzyme A (acetyl-CoA) synthase (ACS) enzyme complex. The methyl group is subsequently reduced to methane by electrons derived from oxidation of the carbonyl group to CO 2 by the CODH-ACS complex (7). An iron-sulfur flavoprotein (Isf) from M. thermophila has been overproduced in Escherichia coli, purified, and characterized. Isf is purified as a homodimer which binds two 4Fe-4S clusters and two flavin mononucleotide (FMN) cofactors (2, 14). Ferredoxin, the electron acceptor of the CODH-ACS complex, is the physiological electron donor for Isf. Additional results support the hypothesis that Isf plays a role in electron transport during fermentation of acetate to methane (14). The deduced amino acid sequence of Isf contains an unusually compact cysteine motif (CX 2 CX 2 CX 5 C) that has been shown, on the basis of site-directed mutagenesis and electron paramagnetic resonance studies, to ligate the 4Fe-4S cluster (15). The midpoint potential values are -394 mV for the 4Fe-4S cluster and -277 mV for the FMN in Isf, suggesting that electrons derived from oxidation of the carbonyl group of acetate flow from ferredoxin to t...

Flavin Mononucleotide-Binding Flavoprotein Family in the Domain Archaea

“…), and desalted with a PD-10 column (Amersham Biosciences AB, Uppsala, Sweden). The purified AfpA was reconstituted with FMN as described previously (32) except that 50 mM HEPES (pH 7.2) was used.…”

“…The isf-2 (AF1519) homolog is one of three tightly clustered genes oriented in the same direction, consistent with an operon (32). Remarkably, an isf homolog (MJ0731) in the genomic sequence of Methanocaldococcus jannaschii (Methanococcus jannaschii) is closely flanked by MJ0732 and MJ0730 with deduced sequences having 46 and 50% identity to the putative proteins encoded by AF1520 and AF1518 that flank isf-2 in A. fulgidus (32). The M. jannaschii open reading frames (ORFs) MJ0732-MJ0731-MJ0730 are all oriented in the same direction.…”

“…Isf (for iron-sulfur flavoprotein) is an electron transfer protein first discovered in Methanosarcina thermophila and is the prototype of a family which includes homologs found in diverse anaerobic species representing all three domains (32). A striking feature of the family is the almost universal occurrence, in individual species, of multiple isf homologs.…”

Structures of the Iron-Sulfur Flavoproteins fromMethanosarcina thermophilaandArchaeoglobus fulgidus

“…The ISF (iron-sulfur flavoprotein) family is distinct from other flavin mononucleotide (FMN)-containing flavoproteins based on overall sequence identity and an unusually compact cysteine motif (24). The prototype of ISF was characterized from Methanosarcina thermophila (ISF-Mt), a strictly anaerobic methane-producing thermophile belonging to the domain Archaea (16).…”

“…The prototype of ISF was characterized from Methanosarcina thermophila (ISF-Mt), a strictly anaerobic methane-producing thermophile belonging to the domain Archaea (16). Since then, homologs of ISF have been identified in the genomes of numerous anaerobic prokaryotes belonging to the domains Bacteria and Archaea (24). Remarkably, many species contain multiple ISF homologs; indeed, the Methanosarcina acetivorans genome is annotated with 19 homologs (12).…”