Nitrogen-15 nuclear magnetic resonance investigation of nitrite reductase-substrate interaction

Nitrogen-15 nuclear magnetic resonance (15N NMR) spectroscopy at 30.4 MHz was employed to determine the interaction of the substrate nitrite (97.2% enriched) with bacterial nitrite reductase, denoted cytochrome cd1, from Pseudomonas aeruginosa. The addition of ferric enzyme to nitrite did not alter the chemical shift of the bulk nitrite resonance, nor was it possible to observe a new resonance from a hypothetical bound form. However, the spin-lattice relaxation time (T1) was lowered from 13.2 to 2.7 s, and the spin-spin relaxation time (T2) was halved. Values of T1 were measured by progressive saturation and values of T2 by line widths. Control experiments involving ferric cytochrome c and metmyoglobin demonstrated that the perturbations did not arise from the bulk paramagnetic properties of the protein solutions. Variable enzyme/substrate ratios were measured to assess the strength of interaction. The most reasonable model consistent with the data proposes a weak association between nitrite and ferric reductase with a value of 1.3 M-1 for the association constant.