BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 224, 611–618 (1996) ARTICLE NO. 1074 Nitrite Reductase from Desulfovibrio desulfuricans (ATCC 27774)— A Heterooligomer Heme Protein with Sulfite Reductase Activity Ine ˆs C. Pereira,* Isabel A. Abreu,* Anto ´nio V. Xavier,* Jean LeGall,* , ² and Miguel Teixeira* ,1 *Instituto de Tecnologia Quı B mica e Biolo ´gica, Universidade Nova de Lisboa, APT 127, 2780 Oeiras, Portugal; and ² Department of Biochemistry, University of Georgia, Athens, Georgia 30622 Received May 15, 1996 The membrane bound cytochrome c nitrite reductase from the sulfate reducer Desulfovibrio desulfuricans (ATCC 27774) was found to have a high specific activity in the reduction of sulfite, producing stoichiometric amounts of sulfide. The K m for sulfite in the MV /l :sulfite oxidoreductase assay is 0.75 mM, and the specific activity 2.06 mmolH 2 /min/mg. Visible and EPR spectroscopies studies indicate that the enzyme high-spin heme reacts with sulfite in the oxidised state, and that sulfide partially reduces the enzyme. The redox- cycled enzyme, using H 2 /Hydrogenase/MV /l as a reductant, is identical to the resting enzyme. This is the first time that a c-type nitrite reductase has been shown to reduce sulfite. These findings, besides revealing a new function for the nitrite reductase, raise a major question regarding the sulfur metabolism in the sulfate reducing bacteria as well as the cellular localization of the enzymatic activities involved in the dissimilatory reduction of sulfate. The purified nitrite reductase is a heterooligomer, containing two types of subunits of 62 kDa ({ 5kDa) and 18.8 kDa ({ 1kDa), and forms a complex or aggregate with a molecular mass of approximately 750 kDa.  1996 Academic Press, Inc. The dissimilatory reduction of sulfate in Desulfovibrio involves essentially three steps: the activation of sulfate to adenylylphosphosulfate (APS), performed by an ATP sulfurylase, the reduction of sulfate to sulfite by APSreductase and finally the reduction of sulfite to sulfide, by the dissimilatory sulfite reductases (1). A major uncertainty resides still on the last step, as it is not clear whether sulfite is reduced in a 6-electron step to sulfide, or if intermediates like trithionate and thiosulfate are involved (2). Although it has generally been assumed that the whole process occurs in the cytoplasm, it has been also proposed that the cytoplasmic membranes may play a role in this pathway (2). Recently, the isolation of desulfoviridin from the membranes of D.desulfuricans (Essex 6) has also been reported (3). This membrane-bound desulfoviridin is identical to the soluble enzyme from the same bacterium except from the fact that it seems to react directly with the tetraheme cytochrome c 3 . Some sulfate reducers have the capacity of using as final electron acceptors other small inorganic ions, such as nitrate and nitrite. In particular, several strains are capable of reducing nitrate to ammonia in a dissimilatory pathway, which involves a nitrate reductase (containing an iron-sulfur center and molybdenum) (4) and a nitrite reductase (NiR) (5). This nitrite reductase is considered as membrane-bound (5,6), and has been proposed to be associated with energy conservation in Desulfovibrio (7,8). It was first described as a monomeric cytochrome containing six c-type hemes (5), five being low-spin and one high-spin in the as-isolated state (9). Similar enzymes have been isolated from other bacteria which perform the dissimilatory reduction of nitrite, as Wolinella succinogenes (10), Escherichia coli (11), two Vibrio species (12,13) and ‘‘Spirillum’’ strain 5175 (14). 1 To whom correspondence should be addressed. 0006-291X/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved. 611