Activation of Nitrite Reductase in a Cell-Free System from the Denitrifier Alcaligenes sp. by Freeze-Thawing

Nitrite reductase (NiR) activity of the cell-free extract orthe soluble fraction prepared from cells of Alcaligenes sp.NC1B 11015 grown anaerobically in the presence of nitrate wasexamined by measuring the rate of nitrite disappearance withdithionitemethyl viologen (MV) as an electron donor. Freezingat — 20?C and subsequent thawing of the fraction resultedin 5-40 times increase of the specific activity of NiR. Fromthe experiments on the effect of freezing conditions on theactivation, the phase change of solvent water due to freezingis considered to play an important role in the activation. Thisactivation occurred with the preparation in the exponentialgrowth phase, but not that in the stationary growth phase. Clearly,the low-molecular-weight (< 12,000) component which was obtainedfrom the soluble fraction through a collodion bag participatedin the activation. The activated enzyme proved to be the dissimilatory NiR, becauseNO production from nitrite, one of the typical characteristicsof the dissimilatory NiR, was also activated when assayed withascorbate-tetramethyl-p-phenylene diamine (TMPD) as an electrondonor. Nevertheless, the reaction products of nitrite reductionwere identified as hydroxylamine and ammonia with dithionite-MV.The possible pathway of nitrite reduction with this electrondonor is discussed. (Received May 26, 1983; Accepted February 2, 1984)