PRIMARY SEQUENCE AND EVIDENCE FOR A PHYSIOLOGICAL-FUNCTION OF THE FLAVOHEMOPROTEIN OF ALCALIGENES-EUTROPHUS

The flavohemoprotein (FHP) encoding gene of the strictly respiratory G ram-negative bacterium Alcaligenes eutrophus was isolated from a megap lasmid library by using FHP-specific antibodies and oligonucleotide pr obes based on the amino-terminal polypeptide sequence of FHP, determin ed previously (Zhu, H., and Riggs, A. F. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5015-5019). The fhp gene codes for a monomeric polypepti de of 403 amino acids (M(r) 44,796) whose structure is highly homologo us to the proteins of the two-domain flavohemoglobin family comprising the hemoproteins from Escherichia coli and Saccharomyces cerevisiae. FHP consists of an amino-terminal-located O-2-binding hemoglobin domai n and a carboxyl-terminal-located redox active domain with potential b inding sites for NAD(P)H and FAD. Two potential binding motifs for NAR L and FNR upstream of fhp suggest a role of FHP in the anaerobic metab olism of A. eutrophus. Isogenic Fhp-negative mutants showed no signifi cant delay in aerobic or anaerobic growth. Compared with the wild type , however, the mutant did not accumulate nitrous oxide during denitrif ication with nitrite as electron acceptor. This property was restored by complementation. The result suggests that FHP interacts directly or indirectly with the gas metabolism during denitrification in A. eutro phus.