Seeing an Enzyme at Work: Conformational Changes Occurring Upon Nitrite Reduction

Nitrite reductase (NiR) is a key enzyme of the denitrification pathway. It catalyses the reduction of nitrite (NO2­) to nitric oxide (NO) according to the reaction:

NO2­ + 2H+ + e­ NO + H2O.

NiR controls the rate of the production of toxic nitric oxide, and regulates its release into the atmosphere. Due to environmental issues, it is crucial to understand the reaction mechanism of such enzymes. Nitrite reductase from the pathogenic Pseudomonas aeruginosa, a soluble 120 kD homodimer, has been studied. The three-dimensional crystallographic structure of the fully oxidised enzyme was solved by molecular replacement from data collected on beamline ID9 [1]. The data were reduced with a new integration package allowing the deconvolution of spatially overlapped diffraction spots, and improving the evaluation of weak spots [2].

As can be seen in Figure 19, each monomer carries a c-domain displaying a classical c-type cytochrome fold and containing a c-heme, and a d1-domain displaying an eight bladed ß-propeller structure and containing a non-covalently bound d1-heme. The c-heme is first reduced in vivo by cytochrome C551, and the electron is slowly transferred to the d1-heme, where the nitrite substrate is reduced. Crystallographic data from the fully reduced enzyme have revealed significant structural modifications relative to the fully oxidised enzyme, an unusual feature in the field of redox enzymes [3]. These modifications appear to be crucial for making the Fe of the d1 heme accessible to the substrate. In order to understand the cascade of events that trigger catalysis, a correlation of the redox-state of the enzyme with its conformational changes was attempted.

A mixed valence state was produced, in which the c-heme is reduced and the d1-heme still oxidised. The experiment was carried out in the following way: starting from the fully oxidised enzyme, ascorbate was rapidly diffused in the crystal, which was then freeze quenched after well-chosen delays. Absorption spectra were recorded for each time-delay on the micro-spectrophotometer (Figure 20). This enabled a crystal of the mixed-valence state to be isolated. This crystal was then exposed to X-rays on ID14. No significant structural modifications relative to the fully oxidised enzyme were observed. Therefore, it was concluded that it was the reduction of the d1-heme that was at the origin of the structural changes observed for the fully reduced enzyme.

This result can be interpreted in the following way: as long as the d1-heme is oxidised, the enzyme needs to prevent binding of the substrate because this would result in a catalytically incompetent complex [1]. It is only once the d1-heme has been reduced that nitrite is allowed to bind. Therefore the structural changes that occur in the fully reduced enzyme, and that relax steric hindrance around the Fe of the d1 heme, must take place after reduction of the latter.

References
[1] N. Nurizzo, M.C. Silvestrini, M. Mathieu, F. Cutruzzola, D. Bourgeois, V. Fülop, J. Hajdu, M. Brunori, M. Tegoni, C. Cambillau, Structure, 5, 1157-1171 (1997).
[2] D. Bourgeois, Acta Cryst. D., D55, 1733-1741 (1999).
[3] N. Nurizzo, F. Cutruzzola, M. Arese, D. Bourgeois, M. Brunori, C. Cambillau, M. Tegoni, J. Biol. Chem, 274-21, 14997-15004 (1999).

Principal Publication and Authors
N. Nurizzo (a), F. Cutruzzola (b), M. Arese (b), D. Bourgeois (c), M. Brunori (b), C. Cambillau (a), M. Tegoni (a), J. Biol. Chem., 274-21, 14997-15004 (1999).

(a) CNRS, AFMB Laboratoire, Marseille (France)
(b) La Sapienza University, Rome (Italy)
(c) IBS/ESRF