Clostridial fermentations Clostridia: physiologically and phylogenically diverse group. 14 related families, at least 7 have organisms traditionally called Clostridium (Collins et al. IJSB 44:812-826, 1994) Genus characteristics: Gram positive Spore formers Butyric acid production.

Clostridia Saccharolytic: swollen cells with subterminal spores C. perfringens group Intestinal organisms Toxins, proteases Sugars or proteins Butyric acid group C. butyricum and C. pasteurianium Solvents by some

Clostridia Tetani: amino acid users Putrefactive group C. tetani Terminal spores Putrefactive group Strictly proteolytic C. sporogenes and C. botulinum Mercaptans, cadaverine, sulfides

Clostridia Substrate-specific groups Acetogens: C. aceticum Purines: C. acidi-urici, C. cylindrosporum Ethanol/acetate: C. kluyveri Acetogens: C. aceticum Make perdominately acetic acid from sugars Some use hydrogen and carbon dioxide to make acetic acid Eubacterium, Peptostreptococcus are intestinal organisms

Idealized clostridial fermentation Glucose Glycolysis 2 ATP (net) 2 NADH 2 Pyruvates 4 H + 2 CoA 2 Fd ox Pyruvate-ferredoxin oxidoreductase 2 Fd red 2 H 2 2 Acetyl-CoA + 2 CO Hydrogenase 2 Acetyl-CoA acetyltransferase CoA O O Acetoacetyl-CoA CH 3 C CH 2 C CoA Β-hydroxybutyryl-CoA dehydrogenase NADH OH O NAD + 3-Hydroxybutyryl-CoA CH 3 CH CH 2 C CoA crotonase O H 2 O Crotonyl-CoA CH 3 CH CH C CoA Butyryl-CoA dehydrogenase NADH NAD + O Butyryl-CoA = CH phosphotransbutyrylase PO 4 3 CH 2 CH 2 C CoA CoA Butyryl-P ATP Yield ADP Butyrate kinase used -2 ATP made 4 + 1 Butyryate Net= 3 ATP/glucose

Claisen condensation to make Acetoacetyl-CoA

Pyruvate and Hydrogen Metabolism Pyruvate:ferredoxin oxidoreductase Contains Thiamine pyrophosphate, But no lipoic acid Oxidative decarboxylation Pyruvate Fdox CoA Fdred CO2 Acetyl-CoA Ferredoxin: small mol. wt., acidic, protein that acts as an electron carrier. Has iron-sulfur redox centers Hydrogen production from pyruvate energetically favorable at any hydrogen partial pressure. Pyruvate- + 2H2O --> acetate - + HCO3 - + 2 H+ + 2 H2 ∆Go’ = -47.3 kJ/mol

Also, used to reoxidize NADH Enzyme: hydrogenase Fdred + 2 H+ --> Fdox + H2 Hydrogen production Uses reduced ferredoxin as electron donor and protons as electron acceptor. Also, used to reoxidize NADH NADH NAD+ Fdox Fdred 2 H+ H2 Hydrogenase NADH:ferredoxin oxidoreductase Net reaction: NADH + 2 H+ --> NAD+ + H2 ∆Go’ = + 18 kJ/mol. Only favorable if pH2 is low.

Fractionation/Reconstitution How do we know that ferredoxin is involved in pyruvate metabolism? 1. Need an assay Cell-free extract + pyruvate = makes H2 - pyruvate = no H2 2. Fractionate and recombine to determine what components are needed to make H2 from pyruvate. Use ion exchange, gel filtration, activated charcoal, etc. to purify. Mortensen et al., 1962, BBRC 7: 448-452 Glass et al. 1977 J. Bact. 131: 463-472

Fractionation of extracts by ion exchange chromatography Apply cell-free extracts Wash with buffer Collect fractions that pass through column. Column packed with ion exchange resin. Proteins with large number of acidic amino acids will stick to column. Collect every 10 ml as a different fraction and assay to see if get hydrogen from pyruvate

Assay results After ion exchange chromatography Hydrogen from pyruvate? DEAE-treated + pyr no H2 - pyr no H2 DEAE-treated with methyl viologen + pyr 6.4 µmol H2 - pyr no H2 Methyl viologen: dye that serves as low potential electron carrier. Conclude: something was removed by chromatography that helps to make hydrogen.

Elution of protein on column with high salt. Apply high salt to remove acidic proteins from column. Find that a brown-colored material elutes from column at 500 mM KCl. Brown material: shown to be protein called ferredoxin

Assay results: After ion exchange chromatography and 500 mM KCl treatment. Hydrogen from pyruvate? DEAE-treated + pyr no H2 - pyr no H2 DEAE-treated with brown material + pyr 6.3 µmol H2 - pyr no H2 Addition of ferredoxin restored activity. Conclude: need ferredoxin to make hydrogen from pyruvate.

Clostridial fermentations In the idealized case, no acetate was formed since all acetyl-CoA was used to make acetoacetyl-CoA to reoxidize NADH In real fermentations, clostridia make a mixture of acetate and butyrate. How can this occur? By having NADH reoxidized by hydrogen production. Only favorable if hydrogen partial pressure is low

Summary Butryic acid formation allows high ATP yield Claisen condensation: common mechanism in biosynthesis using acyl-CoA substrates. Pyruvate:Fd oxidoreductase important enzyme in anaerobic metabolism Hydrogen production from NADH allow fermentative bacteria to maximize ATP gain. Fractionation/reconstitution is how most pathways were elucidated.