Tuomas Haltia - Biochemistry and Biotechnology

Biochemistry of denitrification

Tuomas Haltia, docent, university lecturer

Bacterial denitrification, i.e., stepwise reduction of nitrate into dinitrogen, constitutes an important part of the global nitrogen cycle. Denitrification allows bacteria such as Paracoccus denitrificans to operate their respiratory electron transfer chain and make ATP with a chemiosmotic mechanism even in the absence of oxygen, if nitrate is available. Denitrification occurs in the cell membrane and periplasm of gram-negative microbes and depends on the action of a number of complicated metalloproteins.

Our current research focuses on two denitrification enzymes, namely nitrous oxide reductase (N 2 OR) and nitric oxide reductase (NOR). N 2 OR is a periplasmic copper enzyme which catalyses the reduction of N 2 O into N 2 . It comprises two domains, a cupredoxin domain and a seven-bladed beta-propeller domain (see the Figure below). The cupredoxin domain carries a dinuclear copper centre known as Cu A , whereas another copper centre, a tetranuclear cluster called Cu Z , is located in the propeller domain. The latter forms the site of N 2 O binding and reduction. In contrast, the electron donor proteins of N 2 OR are thought to bind near Cu A , which makes up the electron entry site of the enzyme. A particular question we are stydying deals with the nature of the electron transfer complexes involving either of the two donor proteins of N 2 OR, pseudoazurin and cytochrome c 550 .

NOR reduces two NO molecules to make one N 2 O. It is a two-subunit membrane protein carrying three hemes and a non-heme iron. The large subunit of NOR is related to the catalytic subunit of heme-copper cytochrome oxidases. However, unlike heme-copper oxidases, NOR appears to function electroneutrally. Among other things, this difference makes NOR an attractive object for structural research.

Figure 1.

A nitrous oxide reductase monomer from Paracoccus denitrificans. The cupredoxin domain with the Cu A centre is in upper part of the figure, whereas the seven blades of the propeller form the lower part of the structure. The four coppers of Cu Z and a chloride ion are bound close to the central axis of the propeller. In the cell, two monomers associate to make a head-to-tail dimer.