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-n itrate reductase (EC 1.6.6.1), consisting of the contiguous heme conta ining cytochrome b domain and flavin-containing NADH-cytochrome b redu ctase fragment, has been characterized spectroscopically and kinetical ly. Reductive titration with sodium dithionite indicates heme reductio n takes place prior to flavin reduction, which correlates web 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 sp ectrum of the oxidized fragment is primarily due to the flavin, wherea s the ferrous heme dominates the circular dichroism spectrum of reduce d enzyme. Three kinetic phases are observed in the course of the react ion of the enzyme with NADH, each with a distinct spectral signature. The fast phase represents flavin reduction, concomitant with the forma tion of a charge-transfer complex between reduced flavin and NAD(+), a nd exhibits hyperbolic dependence on NADH concentration with a K-d of 3 mu M and a limiting rate constant of 560 s(-1). Electron transfer fr om reduced flavin to heme with a rate constant of 12 s(-1) is the inte rmediate phase, which is rate-limited by breakdown of the charge-trans fer complex between NAD(+) and reduced flavin. The slow phase is dismu tation of a pair of molecules of two-electron reduced enzyme (generate d 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 o ne-electron reduced enzyme with a second equivalent of NADH to generat e fully reduced enzyme. On the basis of this work, it appears that dis sociation of NAD(+) from the reduced flavin site rate limits electron transfer to the cytochrome and likely represents the overall rate-limi ting step of catalysis.