Poster: Enzymology

Abs # 229: Enzymatic properties of a cyanobcaterial nitrite reductase with a bipartite structure of a [4Fe-4S]/siroheme domain and a [2Fe-2S] ferredoxin domain

Presenter:	Setterdahl, Aaron T., aaron@protein.osaka-u.ac.jp
Authors	Setterdahl, Aaron T. (A)   Hase, Toshiharu  (A)
Affiliations:	(A): Osaka University

Nitrite reductase (NiR), an enzyme containing a single siroheme and an [4Fe-4S] cluster, catalyzes the six-electron reduction of nitrite to ammonia in cyanobacteria and higher plants. Ferredoxin (Fd), an electron carrier containing a single [2Fe-2S] cluster, is the typical electron donor for NiR. Fd sequentially donates six electrons by an intermolecular transfer to the [4Fe-4S] cluster, which then passes the electrons to the siroheme active-site. Plectonema boryanum NiR (pbNiR) is unique in that it contains a redox-active Fd-domain at the C-terminal region of its polypeptide, a property that has not been observed previously in any known sequences of NiR. The NiR domain is homologous to many known assimilatory nitrite reductases from cyanobacteria and higher plants. P. boryanum NiR gene has been cloned from the genomic DNA, ligated into an expression vector and then expressed in Escherichia coli. For comparison, Synechosystis sp. strain 6803 NiR (syNiR) and two truncated versions of pbNiR containing either the NiR domain (ndNiR) or only the Fd-domain were also expressed. Western blot analysis confirmed the presence of full-length NiR in the cell extract of P. boryanum. Both pbNiR and ndNiR are catalytically active with either methyl viologen, P. boryanum Fd (petF), or Fd-domain as electron donors. Authentic NiR purified from P. boryanum has similar characteristics to the recombinant pbNiR. Cyclic voltammetry of Fd-domain shows a higher reduction potential of -260 mV compared with -350 mV for petF. In order to determine the function of the Fd-domain, experiments include characterization of the enzymatic properties of pbNiR, ndNiR, and syNiR, and determination of the reduction potentials of the siroheme and iron-sulfur clusters.