Dissertation submitted to obtain the phD degree
in Biochemistry, specialty in Physical-
Biochemistry, by the Faculdade de Ciências e
Tecnologia from the Universidade Nova de
LisboaDenitrification is a metabolic pathway from the nitrogen cycle where nitrate is reduced
to dinitrogen. This pathway involves four reduction steps: the nitrate reduction to nitrite, followed by the reduction to nitric oxide (NO), from this to nitrous oxide (N2O), and finally to dinitrogen. The enzymes involved in the mentioned steps are the nitrate reductases, the nitrite reductases, the nitric oxide reductases (NOR) and the nitrous oxide reductases, respectively.
The NORs perform the NO reduction to N2O, using two substrate molecules, two protons and two electrons with the consequent product formation and water. These enzymes belong to the heme copper oxidase superfamily, since they are integral
membrane proteins, with 12 transmembrane α-helixes and a set of conserved residues.
They are composed by two subunits. The NorC subunit with a molecular weight of 17
kDa, comprises a low-spin heme c covalently bound to the polypeptide chain, with a
His/Met coordination. The second subunit, NorB, also named the catalytic subunit, with
a molecular weight of 56 kDa, harbouring two b-type hemes, one low-spin bis-His
coordinated (heme b), a high-spin heme b (heme b3), His coordinated and a non-heme
FeB. These last two iron centers are antiferromagnetic coupled and bridged by a μ-
oxo/hydroxo group, and together they compose the catalytic diiron center. Recently
Mössbauer spectroscopy proved that the catalytic heme b3 is in fact low-spin in both
ferric and ferrous states, indicating a six-coordination environment for this iron center in both redox states. The Pseudomonas (Ps.) aeruginosa NOR crystal structure shows the presence of the His ligand simultaneously with the μ-oxo bridge in the as-isolated form.
The substrate reduction mechanism is an issue of intense discussion, with the cis and
trans-mechanisms taken in consideration. The cis-mechanism descries that NO reduction
occurs in the catalytic center and only one of the iron atoms is coordinating the substrate during catalysis. The trans-mechanism describes NO reduction with the binding of one substrate molecule to each one of the iron atoms of the catalytic center. Different isolated NORs have the ability of reducing O2 to H2O in a four electrons/four protons reaction.
The mechanism for O2 reduction is unknown, but it is presumed that substrate binds to
the catalytic heme b3, analogous to the terminal oxidases. The main objective of this work was to isolate and characterize the Ps. nautica NOR, using different biochemical, and spectroscopic techniques.Fundação para a Ciência e Tecnologia - bolsa de doutoramento (SFRH/BD/39009/2007
