Alternate pathways for NADH oxidation in Thermus thermophilus using type 2 NADH dehydrogenases

Type 2 NADH dehydrogenase (NDH-2) is a single- subunit membrane-associated flavoenzyme that is part of the respiratory chain of many prokaryotes. The enzyme catalyzes the electron transfer from NADH to quinone but is not directly coupled to the generation of a proton motive force. The purpose of the current work is to compare two different NDH-2s that are encoded in strains of Thermus thermophilus. The aerobic T. thermophilus HB27 strain expresses one NDH-2 that has been previously isolated and characterized. In this work it is shown that a gene, which is misannotated as an NADH oxidase, encodes this enzyme. Unlike HB27, strain NAR1 of T. thermophilus is capable of partial denitrification, and in addition its genome contains the nrcN gene that encodes a second putative NDH-2. Of particular interest is the fact that nrcN is part of an operon ( nrcDEFN ) that is proposed to encode a protein complex specifically required for nitrate reduction. In this work, the nrcN gene has the activity expected of a NDH-2, and functions independently of other components of the putative Nrc complex. The biochemical properties of the two NDH-2 enzymes are compared. Efforts to demonstrate that NrcN is part of a multiprotein complex were not successful. However, the NrcE protein was expressed in Escherichia coli and shown to be a membrane-bound protein containing heme B.