1. Denitrification by Pyrobaculum aerophilum.

P. aerophilum is a marine archaeon that grows optimally at 100ºC. It is one of the few facultative anaerobic hyperthermophilic archaea isolated thus far that can grow with nitrate as electron acceptor. Nitrate is reduced via the denitrification pathway to N2 gas. Denitrification is of fundamental importance for the balance of the global nitrogen cycle since it is the main biological process that converts fixed nitrogen compounds back to N2.

Our initial experiments led to the discovery that P. aerophilum is strictly dependent on the presence of tungsten in the culture medium for cell growth with nitrate. Tungsten is found in the natural environment of hyperthermophiles such as P. aerophilum, however, it is usually absent in cooler environments. Because of its antagonistic nature, tungsten interferes with the molybdenum metabolism of most organisms in nature. Our goal is to understand how P. aerophilum regulates its tungsten and molybdenum-dependent metabolism.

To understand the involvement of metal cofactors and to compare mesophilic, bacterial denitrifiers to hyperthermophilic Archaea, our goal is to purify all enzymes involved in the denitrification pathway of P. aerophilum. In contrast to bacteria, all four denitrification enzymes of P. aerophilum are all located in the cytoplasmic membrane. In addition, we are in the process of elucidating the enzymes that serve as the electron donor to the nitrate respiratory enzymes and to characterize the electron-mediating quinone.

2. Ferric reduction by Archaeoglobus fulgidus.

A. fulgidus is a marine, strict anaerobic archaeon with a growth optimum temperature of 83ºC. A. fulgidus utilizes a variety of organic and inorganic compounds as electron donor but is limited to sulfate as the electron acceptor for growth.

Assimilatory iron metabolism is a fundamental process in all living organisms, however, no information about this process in the archaea exists. We have recently isolated a ferric reductase enzyme from the soluble protein fraction of this organism. This enzyme reduces complexed ferric iron (Fe3+) to ferrous iron (Fe2+) using NAD(P)H as the electron donor. The ferric reductase was characterized biochemically and represents the first archaeal ferric reductase that has been described thus far. The A. fulgidus enzyme is distinct from eukaryotic assimilatory type ferric reductase and has some resemblance to the bacterial enzymes. We have cloned the A. fulgidus ferric reductase, and the recombinant enzyme was crystallized. The structure was solved of a complex with FMN, which acts a cofactor and as a complex with both FMN and the electron donor NADPH. The A. fulgidus ferric reductase is unique in that it is the first enzyme identified that coordinates NADP without a Rossman fold domain. Our goal is to elucidate the function of A. fulgidus genes that form an apparent transcriptional unit with the ferric reductase gene. This should give insight into how iron is assimilated in the archaea.