KINETIC STUDIES ON CYTOCHROME cd₁ NITRITE REDUCTASE. P.A. Williams, V. Fülöp, E.F. Garman, and J. Hajdu. Laboratory of Molecular Biophysics, and Oxford Centre of Molecular Sciences, University of Oxford, UK

Use of cryo-crystallography and spectroscopic techniques has enabled the determination of intermediate substrate and product complexes on cytochrome cd1 nitrite reductase during the reduction of nitrite.

Cytochrome cd₁ nitrie reductase is a dimeric enzyme, consisting of a small cytochrome domain containing a c haem, and a larger beta-propeller domain containing a non-covalently attached d₁ haem. The crystal structure of the fully oxidised form of cytochome cd₁ has previously been published at a resolution of 1.55Å (1). The structure of the fully reduced form of the enzyme has also been determined to a resolution of 2.0Å (Williams et al, unpublished).

The enzyme is active in the crystalline form, and the reaction can be followed using a microspectrophotometer (2). There are several distinct spectroscopic intermediates, with lifetimes of 10 seconds - 5 minutes. The crystals proved unsuitable for Laue analysis, and thus structural intermediates were trapped in the crystal by shock-cooling reacting crystals to 100K. Several points on the catalytic pathway have been determined, giving insights into the mechanism of nitric oxide release from cytochrome cd₁.

(1) Fülöp, V., Moir, J.W.B., Ferguson, S.J. and Hajdu, J. (1995). Cell 81, 369-377

(2) Hadfield, A. and Hajdu, J. (1993). J. Appl. Cryst. 26 839-842