Spectroscopic and kinetic characterization of the recombinant cytochrome c reductase fragment of nitrate reductase. Identification of the rate-limiting catalytic step.

The recombinant NADH-cytochrome c reductase fragment of spinach NADH-nitrate reductase (EC 1.6.6.1), consisting of the contiguous heme-containing cytochrome b domain and flavin-containing NADH-cytochrome b reductase fragment, has been characterized spectroscopically and kinetically. Reductive titration with sodium dithionite indicates heme reduction takes place prior to flavin reduction, which correlates well with the reduction potentials for enzyme-bound heme (15 mV) and FAD (-280 mV). Reductive titration with NADH also indicates that the reduced enzyme forms a charge-transfer complex with NAD+. The circular dichroism spectrum of the oxidized fragment is primarily due to the flavin, whereas the ferrous heme dominates the circular dichroism spectrum of reduced enzyme. Three kinetic phases are observed in the course of the reaction of the enzyme with NADH, each with a distinct spectral signature. The fast phase represents flavin reduction, concomitant with the formation of a charge-transfer complex between reduced flavin and NAD+, and exhibits hyperbolic dependence on NADH concentration with a Kd of 3 microM and a limiting rate constant of 560 s-1. Electron transfer from reduced flavin to heme with a rate constant of 12 s-1 is the intermediate phase, which is rate-limited by breakdown of the charge-transfer complex between NAD+ and reduced flavin. The slow phase is dismutation of a pair of molecules of two-electron reduced enzyme (generated at the end of the second phase of the reaction) to give one molecule each of one- and three- electron reduced enzyme, with a second order rate constant of 2 x 10(6) M-1 s-1. In the presence of excess NADH, this dismutation reaction is followed by the rapid reaction of the one-electron reduced enzyme with a second equivalent of NADH to generate fully reduced enzyme. On the basis of this work, it appears that dissociation of NAD+ from the reduced flavin site rate limits electron transfer to the cytochrome and likely represents the overall rate-limiting step of catalysis.