The structurally characterized flavohemoprotein from Alcaligenes eutrophus (FHP) contains a phospholipidbinding site with 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-ethanolamine and 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-glycerol as the major occupying compounds. The structure of the phospholipid is characterized by its compact form, due to the -sc/b/-sc conformation of the glycerol and the nonlinear arrangement of the sn-1-and sn-2-fatty acid chains. The phospholipid-binding site is located adjacent to the heme molecule at the bottom of a large cavity. The fatty acid chains form a large number of van der Waal's contacts with nonpolar side chains, whereas the glycerophosphate moiety, which points towards the entrance of the channel, is linked to the protein matrix by polar interactions. The thermodynamically stable globin module of FHP, obtained after cleaving off the oxidoreductase module, also contains the phospholipid and can therefore be considered as a phospholipidbinding protein. Single amino acid exchanges designed to decrease the lipid-binding site revealed both the possibility of blocking incorporation of the phospholipid and its capability to evade steric barriers. Conformational changes in the phospholipid can also be induced by binding heme-ligating compounds. Phospholipid binding is not a general feature of flavohemoproteins, because the Escherichia coli and the yeast protein exhibit less and no lipid affinity, respectively.Keywords: crystal structure; flavohemoprotein; globin; phospholipid; protein engineering.Flavohemoprotein is a monomeric protein with a molecular mass of 43 kDa, which binds one heme (Fe-protoporphyrine IX) and one FAD as prostethic groups and NADH as a cofactor. This protein has been identified and characterized in an expanding number of prokaryotic and eukaryotic organisms, comprising Flavohemoprotein was originally isolated from the gramnegative bacterium A. eutrophus (denoted as FHP) [11] and characterized with respect to its spectral properties [12], its primary structure [1] and its crystal structure [13]. According to a 0.175-nm structure, FHP can be subdivided into three domains. The N-terminal 147 residues comprise the well-known globin fold[14] composed of seven a helices forming a hydrophobic crevice, where the heme molecule is embedded. The FAD-binding domain (105 residues) basically consists of a six-stranded antiparallel b barrel surrounded by a helix and an irregular peptide segment. The noncovalently bound FAD binds roughly to the face of the b sheet. The architecture of the NAD-binding domain (138 residues) corresponds to a Rossmann fold composed of a five-stranded parallel b sheet flanked by two helices and an irregular structural element. The binding mode of NADH is not yet established. The FAD-binding and NAD-binding domains are fused to the so-called oxidoreductase module, which structurally belongs to the ferredoxin reductase family [15]. FHP is therefore composed of two essentially independent globin and oxidoreductase modules.Lipid-binding protei...