Transition Metals in Biochemistry 1155 New insights into the activity of Pseudomonas aeruginosa cd 1 nitrite reductase Serena Rinaldo*, Alessandro Arcovito†, Giorgio Giardina*, Nicoletta Castiglione*, Maurizio Brunori* and Francesca Cutruzzol ` a* 1 *Dipartimento di Scienze Biochimiche “A. Rossi Fanelli” and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza, Universit ` a di Roma, Rome, Italy, and †Istituto di Biochimica e Biochimica Clinica, Universit ` a Cattolica del Sacro Cuore, Rome, Italy Abstract The cytochrome cd 1 nitrite reductases are enzymes that catalyse the reduction of nitrite to nitric oxide (NO) in the bacterial energy conversion denitrification process. These enzymes contain two different redox centres: one covalently bound c-haem, which is reduced by external donors, and one peculiar d 1 -haem, where catalysis occurs. In the present paper, we summarize the current understanding of the reaction of nitrite reduction in the light of the most recent results on the enzyme from Pseudomonas aeruginosa and discuss the differences between enzymes from different organisms. We have evidence that release of NO from the ferrous d 1 -haem occurs rapidly enough to be fully compatible with the turnover, in contrast with previous hypotheses, and that the substrate nitrite is able to displace NO from the d 1 -haem iron. These results shed light on the mechanistic details of the activity of cd 1 nitrite reductases and on the biological role of the d 1 -haem, whose presence in this class of enzymes has to date been unexplained. Introduction Pseudomonas aeruginosa is an opportunistic pathogen that is able to colonize low-oxygen environments such as the air- ways of cystic fibrosis patients, where it survives using the anaerobic respiratory pathway of denitrification [1]. In the denitrification process, nitrate is reduced to dinitrogen by four different enzymes (namely nitrate, nitrite, nitric oxide and nitrous oxide reductases) [2], according to the following pathway: NO − 3 → NO − 2 → NO → N 2 O → N 2 This pathway is expressed under low oxygen tension in the presence of N-oxides such as nitrate and nitrite [2]. Intracellular NO concentration is strictly controlled by the co-ordinated activity of the enzymes nitrite and nitric oxide reductases, which catalyse NO production and removal respectively [1,2]. The NiR (nitrite reductase) from Ps. aeruginosa (Pa- cd 1 NiR) is a homodimer containing in each monomer one covalently bound c-haem, which is reduced by external donors, and one d 1 -haem, where catalysis occurs [3]. The d 1 -haem, a cofactor unique to the denitrifiers containing the cd 1 NiR, is synthesized starting from δ-aminolevulinic acid via uroporphyrinogen III. The d 1 -haem, which was unequi- vocally identified as a 3,8-dioxo-17-acrylate-porphyrindione (Figure 1), has also been confirmed by inspection of the crystal structures of cd 1 NiRs [4,5]. The presence of the electronegative oxo groups shifts the redox potential ( E ′ 0 ) of Key words: cytochrome cd1, haem d1, nitric oxide, nitrite reductase, Pseudomonas aeruginosa. Abbreviations used: NiR, nitrite reductase; Pa-cd1NiR, Pseudomonas aeruginosa cd1 NiR; Pp-cd1NiR, Paracoccus pantotrophus cd1NiR. 1 To whom correspondence should be addressed (email francesca.cutruzzola@ uniroma1.it). the iron to more positive values, relative to protoporphy- rin IX. EPR, NMR and MCD (magnetic circular dichroism) measurements [6–8] showed that the d 1 -haem in Pa-cd 1 NiR is a low-spin hexa-co-ordinate species in the ferric state and a high-spin pentaco-ordinate species in the ferrous state. The so-called proximal ligand is provided by His 182 . In the low-spin ferric form, the sixth ligand is a hydroxide ion in the oxidized Pa-cd 1 NiR structure. However, in the oxid- ized Paracoccus pantotrophus cd 1 NiR (Pp-cd 1 NiR), the sixth ligand is provided by the phenolate of Tyr 25 , a residue be- longing to the N-terminal segment and thus to the c-domain; this state is catalytically inert (resting) and needs to be activated by reduction [9,10]. A low-spin ferric d 1 -haem- associated α-band in the 636–644 nm region, seen in all NiRs, may be consistent with a common histidine–OH or histidine– tyrosine ligation; however, whether the latter form is on or off the catalytic pathway remains unclear. Two conserved histidine residues (His 327 and His 369 ) are present in the distal pocket of the d 1 -haem in Pa-cd 1 NiR and are both involved in catalysis (see below) [11]. Reduction of nitrite: substrate binding and product release In vitro, Pa-cd 1 NiR catalyses both the reduction of nitrite to NO and the reduction of dioxygen to water. Reduced d 1 - haem binds the oxidants (NO 2 − ,O 2 ) as well as other haem ligands, such as NO, carbon monoxide (CO) and cyanide (CN − ). Nitrite reduction is, however, the physiologically relevant activity of the enzyme; reduced Pa-cd 1 NiR binds nitrite with high affinity (K m = 6 μM) [11] and produces NO with a turnover number of 6 s −1 (at pH 7.0) [12]. The activity Biochem. Soc. Trans. (2008) 36, 1155–1159; doi:10.1042/BST0361155 C  The Authors Journal compilation C  2008 Biochemical Society Biochemical Society Transactions www.biochemsoctrans.org