Purification and Characterization of a Ferredoxin from Acetate-grown Methanosarcina thermophila*

A ferredoxin, which functions as an electron accep- tor for the CO dehydrogenase complex from Methanosarcina thermophila, was purified from acetate- grown cells. It was isolated as a trimer having a native molecular weight of approximately 16,400 and mon- omer molecular weight of 4,888 calculated from the amino acid composition. The ferredoxin contained 2.80 .C 0.56 Fe atoms and 1.98 r?: 0.12 acid-labile sulfide. UV-visible absorption maxima were 395 and 295 nm with monomeric extinction coefficients of essc = 12,800 M" cm" and e296 = 14,460 M" cm". The A898/A295 ratio ranged from 0.80 to 0.88. There were 5 cysteines per monomer but no methionine, histidine, arginine, or aromatic amino acids. The N-terminal amino acid sequence showed a 4-cysteine cluster with potential to coordinate a Fe:S center. The protein was stable for 30 min at 70 "C, but denatured during incubation at 85 "C. M e ~ ~ ~ ~ o s u r c ~ n a t ~ r ~ o p ~ i ~ is an acetotrophic methane-producing archaebacterium. During the dissimilation of acetate to methane, it is hypothesized that a multi-enzyme complex with CO dehydrogenase activity catalyzes carbon-carbon bond cleavage of acetyl-coA (1, 2). It has been proposed that the carbonyl

All steps were performed anaerobically at 23 "C in a Coy anaerobic chamber (Coy Laboratory Products, Ann Arbor, MI). Soluble components of cell extract (130 mg of protein) were ultrafiltered in an Amicon pressure dialysis apparatus (Amicon Corp., Lexington, MA) equipped with a YM-30 membrane. The retentate was washed with 3 volumes (60 ml) of buffer. (Although the native molecular weight is less than 30,000, the ferredoxin did not pass through the membrane.) The retentate (117 mg) was then fractionated by fast protein liquid chromatography using a model GP-250 gradient programmer (Pharmacia LKB Biotechnology Inc.) and a Mono-Q HR 10/10 anion exchange column (Pharmacia LKB Biotechnology Inc.). The column was developed with a linear gradient of 0.0-1.0 M KCl. Ferredoxin were pooled (3.2 mg of protein), diluted with buffer, and further was the last major protein to elute from the column; active fractions purified by batch elution from the Mono-Q column with 0.49 M KC1. As a final purification step, Mono-Q fractions containing ferredoxin (3.1 mg) were chromatographed using a Superose-12 (Pharmacia LKB Biotechnology Inc.) gel filtration column (M, range = 1,000-300,000).
Pure ferredoxin (2.8 mg) was stored in liquid N, until use.
Spectroscopy-UV-visible absorption spectra were obtained at 23 "C using a Perkin-Elmer Lambda 4B spectrophotometer and the Spectral Processing operating software package. The thermal stability of ferredoxin was monitored by following the decrease in absorbance at 400 nm during incubation at 70 or 85 "C (7).
Amino Acid Composition and N-terminal Amino Acid Analysis-Ferredoxin samples were oxidized with performic acid for 4 h at 4 "C by the method of Moore (8) and hydrolyzed in uacuo with 6 N HC1 at 115 "C for 24, 48, and 72 h. Amino acids were analyzed using Dionex Analyzers (Dionex Corp., Sunnyvale, CA), models D-500 and D-502.
Tryptophan was not determined.
N-terminal amino acids were sequenced using an Applied Biosystems (Applied Biosystems, Inc., Foster City, CA) 470 A gas phase peptide sequencer and identifying the phenylthiohydantoin derivative (9) with an on-line Applied Biosystems liquid chromatograph.
Inorganic sulfide was determined by the micro method of Beinert (10) in 0.5-ml culture tubes fitted with serum stoppers (7 X 15 mm). Iron was determined by atomic absorption spectroscopy with a Perkin-Elmer atomic absorption s~c t r o p h o t o m e~r model 560. A standard curve was calibrated using dilutions of a certified atomic absorption iron standard (Fisher). Ferredoxin solutions were maintained anaerobic until analyzed.
Protein concentrations of ferredoxin preparations were determined by the total weight of amino acids released during hydrolysis and compared to the values obtained by the micro method of Pierce  (Pierce Chemical Go.) using bicinchoninic acid protein assay reagent. A correction factor which included differences in the average residue molecular weight between ferredoxin and bovine serum albumin was employed in subsequent protein determinations by the Pierce method. All other protein concentrations were determined by the method of Bradford (11) using Bio-Rad protein dye reagent (Bio-Rad) and bovine serum albumin as standard.

4080
Chem~u~-Bovine serum albumin (fraction V), methyl viologea, MES, 2-mercapt~~hanol (type I), TES, and ~~-~e t h y l -~-p h e nylenediamine m o n o h y~~h l o r i d e were obtained from Sigma. Sucrose (reagent grade) was obtained from Fisher.

RESULTS
Physical and Chemical Properties-Ferredoxin was purified anaerobically from soluble proteins of acetate-grown M . thermophila. N-terminal amino acid sequence analysis revealed only one peptide chain. The native molecular weight, estimated by Sephadex G-50 gel filtration chromatography, was 16,400. The minimum molecular weight, calculated from the amino acid composition (Table I) was 4,888, suggesting that the ferredoxin was isolated as a trimer, Analysis by atomic absorption spectroscopy showed 2.80 rt 0.46 ( n = 7) Fe/moI of monomer. The acid-Iabile sulfide content was 1.98 rt 0.12 ( n = 5) S/mol. Estimations of acid-labile sulfide were consistently lower than those for iron which probably resulted from losses of sulfide during analysis. Assuming an underestimation of the acid-labile sulfide atoms, these results are consistent with one 3Fe:3S co~/monomer. Spectral Properties-The UV-visible absorption spectrum of oxidized ferredoxin showed broad bands centered at 395 and 295 nm (Fig. 1). The ferredoxin spectrum was stable in air for 60 min at 23 "C. The ratio of A395/A29b ranged from 0.8 to 0.88 which is typical of pure ferredoxins and further supports the purity of the preparation. Based on a molecular weight of 4,888, the extinction coefficients at 295 and 395 nm were 14,460 and 12,800 M" cm", respectively. The extinction coefficient at 395 nm is similar to the 1 X 3Fe:3S ~e s~l~o u~~r~ gigas ferredoxin 11 molar absorption coefficient at 415 nm of Following acidification of the ferredoxin solution with HCI to pH 3, the absorbance decreased in the 395 and 295 nm region indicating destruction of the Fe:S core (Fig. 1). Reduction with dithionite resulted in decreased absorbance in the 15,700 M" cm-l (12). ~erredoxin from Aceta~e-g~own ~e~~~s a r~i~ . No other chromophores were unmasked following acidification or reduction. Table I shows the total amino acid composition of ferredoxin from acetate-grown M. thermophila. Methionine, histidine, arginine, and aromatic amino acids were absent, as was reported for ferredoxins isolated from two strains of methanol-grown Methanosarcina barkeri (Table I). The minimum molecular weight of the M. thermophila ferredoxin, determined from the amino acid composition, was 4,888. The protein contained 5 cysteines/monomer and a preponderance of acidic and hydrophobic amino acids. N-terminal sequence analysis of purified ferredoxin revealed only one polypeptide chain. The first 39 amino acids are compared with the N-terminal sequence of 3Fe:3S and 4Fe:4S ferredoxins from methanogens and eubacteria (Fig. 2).

Amino Acid Composition and N-terminal Analysis-
Thermal Stability-C. posteurianum ferredoxin rapidly denatured at 70 "C; however, the protein from M. t h e r~p h i l a was relatively stable at 70 "C for 30 min, but rapidly denatured upon incubation at 85 "C (Fig. 3). Ferredoxin preparations were active when assayed after 15 min at 70 "C, but inactive following incubation for 15 min above 80 "C (data not shown).

DISCUSSION
In addition to the ferredoxin of M. thermophila reported here, two others have been described in two methanol-grown strains of M. barkeri (13,14). The ferredoxin from M. barkeri (DSM 804) (14), which couples Hz evolution and the pyruvate dehydrogenase system, has been characterized and appears to be different from the ferredoxin described in this work based on the following: (i) the iron and acid-labile sulfide content of the M. thermophila ferredoxin is approximately half of that obtained for the M. barkeri (DSM 804) protein, (ii) the extinction coefficient at 410 nm of 36,500 M" cm" obtained for M. burkeri (DSM 804) ferredoxin (14) is twice the value obtained for the M. t h e r~p h i l a protein, and (iii) the M. thermophila ferredoxin contains 48 amino acid residues and only 5 cysteines/monomer compared to a total of 60 residues and 8 cys~ines/monomer in the M. barkeri (DSM 804) protein (Table I).
The ferredoxin from acetate-grown M. thermophila shares some properties with the ferredoxin purified from methanolgrown M. barkeri (DSM 800). The iron and acid-labile sulfide content, extinction coefficients, thermal stability, or physiological activity of the M. barkeri protein were not reported; however, EPR and Mossbauer spectroscopy indicate a 3Fe:3S core structure (18). Dithionite-reduced M. thermophila ferredoxin was EPR silent; however, air-oxidized samples gave g-  (Table I) although the M. ther~ophila protein contained 5 cysteines/ monomer compared to 8/monomer in M. barkeri (DSM 800) ferredoxin and a total of 48 amino acids compared to 59/ monomer (13). Examination of the sequence of the first 39 N-terminal amino acids showed that the ferredoxins are 85% homologous (Fig. 2). This value is high relative to the 24% DNA homology between M. thermophila and M. barkeri (MS) (19), indicating that the N-terminal sequence is highly consewed between the two species. However, the lower number of cysteine residues in M. thermophila ferredoxin and the fact that both of the prolines from the amino acid composition are present in the N terminus suggests that a second 4-cysteine * K. C. Terlesky and J. G. Ferry, unpublished results. cluster is not possible. Thus, unlike the M. barkeri (DSM 800) protein, it is likely the M . t h e r~p h i l a ferredoxin contains a single 3Fe:3S cluster. The ferredoxin functions as an electron acceptor for CO dehydrogenase complex and hydrogenase which implies that the Fe:S center has a very low redox potential.
The spacing of the cysteines and the overall amino acid homology do not necessarily indicate the type of Fe:S core accommodated (13). Initial findings suggest that M. thermophila ferredoxin contains only a 3Fe:3S core/monomer. Strictly anaerobic purification procedures were used; therefore, it is unlikely conversion of a 4Fe to a 3Fe core occurred during isolation of the protein. However, in vivo interconversion of 4Fe and 3Fe centers (20) cannot be ruled out. Conversely, published results suggest the M. barkeri (DSM 804) ferredoxin coordmates two 4Fe:4S centers (14), although this protein is 85 and 90% homologous with the first 20 amino acids of the M. the~mophila and M. barkeri (DSM 800) proteins.
Azotobacter uinelandii ferredoxin contains the Cl6VEIs tripeptide ( Fig. 2) which is thought to coordinate one Fe of the 3Fe:3S center through the cysteinyl and, possibly, glutamyl side chains (6) barkeri (DSM 800) contain a corresponding tripeptide (C15VD,,), albeit with a conservative substitution for glutamate (Fig. 2). Because the M. thermophila ferredoxin contains only 5 cysteines/monomer, it is tempting to hypothesize that the CisVD17 tripeptide coordinates one Fe atom and that the remaining 4 cysteines coordinate two Fe atoms, as proposed for the A. vinelandii 3Fe:3S center (6); alternatively, the noncysteinyl ligand may be an exogenous molecule such as water. Clearly, more research is needed to determine the structure of the Fe:S center in the M. thermophila ferredoxin.