Alcohol dehydrogenases (ADHs) from thermophilic microorganisms are interesting enzymes that have their potential applications in biotechnology and potentially provide insight into the mechanisms of action of thermo-tolerant proteins. The molecular mechanisms of ADHs under thermal stress in vivo have yet to be explored. Herein, we employed a proteomic strategy to survey the possible interactions of secondary-ADH (2-ADH) with other proteins in Thermoanaerobacter tengcongensis (T. tengcongensis) cultured at 75 C and found that 2-ADH, pyruvate-ferredoxin oxidoreductase (PFOR) and several glycolytic enzymes coexisted in a protein complex. Using anion exchange chromatography, the elution profile indicated that the native 2-ADH was present in two forms, PFOR-bound and PFOR-free. Immuno-precipitation and pull down analysis further validated the interactions between 2-ADH and PFOR. The kinetic behaviours of 2-ADH either in the recombinant or native form were evaluated with different substrates. The enzyme activity of 2-ADH was inhibited in a non-competitive mode by PFOR, implying the interaction of 2-ADH and PFOR negatively regulated alcohol formation. In T. tengcongensis, PFOR is an enzyme complex located at the upstream of 2-ADH in the alcohol generation pathway. These findings, therefore, offered a plausible mechanism for how alcohol metabolism is regulated by hetero-interactions between 2-ADH and PFOR, especially in anaerobic thermophiles. (C) 2011 Elsevier Inc. All rights reserved.