Escherichia coliFlavohemoglobin Is an Efficient Alkylhydroperoxide Reductase

Alessandra Bonamore, Patrizia Gentili, Andrea Ilari, M. Eugenia Schininà, Alberto Boffi
2003 Journal of Biological Chemistry  
Escherichia coli flavohemoglobin (HMP) is shown to be capable of catalyzing the reduction of several alkylhydroperoxide substrates into their corresponding alcohols using NADH as an electron donor. In particular, HMP possesses a high catalytic activity and a low K m toward cumyl, linoleic acid, and tert-butyl hydroperoxides, whereas it is a less efficient hydrogen peroxide scavenger. An analysis of UV-visible spectra during the stationary state reveals that at variance with classical
more » ... , HMP turns over in the ferrous state. In particular, an iron oxygen adduct intermediate whose spectrum is similar to that reported for the oxo-ferryl derivative in peroxidases (Compound II), has been identified during the catalysis of hydrogen peroxide reduction. This finding suggests that hydroperoxide cleavage occurs upon direct binding of a peroxide oxygen atom to the ferrous heme iron. Competitive inhibition of the alkylhydroperoxide reductase activity by carbon monoxide has also been observed, thus confirming that heme iron is directly involved in the catalytic mechanism of hydroperoxide reduction. The alkylhydroperoxide reductase activity taken together with the unique lipid binding properties of HMP suggests that this protein is most likely involved in the repair of the lipid membrane oxidative damage generated during oxidative/nitrosative stress. "Hemoglobin-like" proteins represent an increasingly growing family of globins whose genes are widespread among prokaryotic and eukaryotic microorganisms (1, 2). These proteins are structurally related to vertebrate hemoglobins and myoglobins in that their architecture is based on the typical globin fold and the heme is linked to the polypeptide chain through a proximal histidine residue. Two main classes of hemoglobin-like proteins have been identified, namely single domain bacterial hemoglobins (comprising the so-called truncated hemoglobins) and flavohemoglobins in which the globin domain is fused with a ferredoxin reductase-like FAD and NAD binding module. The "functional annotations" of these proteins are still controversial, and different physiological roles have been proposed that span from simple oxygen delivery to terminal oxidases (facilitated diffusion) to more complex enzymatic activities linked to oxidative and/or nitrosative stress cell responses (1, 2). The flavohemoglobin from Escherichia coli (HMP) 1 has been the object of a large number of investigations to unveil its physiological role in the framework of bacterial resistance to nitrosative stress. HMP expression has been demonstrated to respond to the presence of nitric oxide (NO) in the culture medium, and an explicit mechanism has been proposed that involves NO scavenging and its reduction to N 2 O under anaerobic conditions (3). In contrast to (or together with) the anaerobic NO reductase activity, HMP has also been shown to be able to catalyze the oxidation of free NO to nitrate (nitric-oxide dioxygenase activity) both in vivo and in vitro in the presence of oxygen and NADH (4 -7). Alternatively, an NO denitrosylase function has been proposed in which, at low oxygen tensions, HMP turns over in the ferric state with the intermediacy of an iron-bound nitroxyl anion that is subsequently transformed into nitrate in the presence of oxygen (8). It remains to be established which of these diverse enzymatic activities correspond to a physiologically relevant process. A second set of functional hypotheses has been inferred on the basis of the structural features of the active site of HMP (9) and other closely related proteins. HMP, similar to the single chain hemoglobin from Vitreoscilla sp. (10) and the flavohemoglobin from Alcaligenes eutrophus (11), displays a structural geometry of the active site that is strongly reminiscent of that of typical peroxidases. In particular, the x-ray crystal structures of all of the above mentioned proteins show that the proximal heme pocket is characterized by a network of hydrogen-bonding interactions that comprises the N⑀ atom (hydrogen-bonding donor) of the proximal histidine, thus conferring an anionic character to the imidazole ring (9, 11). This feature allows a clear cut structural distinction between genuine hemoglobins in which the integrity of the ligand in the trans position needs to be preserved and peroxidases in which electron donation to the trans ligand is at the core of the catalytic activity (electron push effect). The spectroscopic signature of the peroxidase-like character of the active site is provided by resonance Raman measurements on the fully reduced, deoxygenated HMP derivative whose spectrum displays a very high iron histidine-stretching frequency, in line with the values observed for typical peroxidases (12). Subsequently, FTIR measurements conducted on the cyanide-bound ferric species confirmed the structural analogies between HMP and peroxidases highlighting the strong electron donation to the ironbound ligand (13). Nevertheless, an extensive screening of possible peroxidase and/or monooxygenase activities toward a number of typical peroxidase and/or cytochrome P-450 sub-
doi:10.1074/jbc.m301285200 pmid:12663656 fatcat:pp4qmrpzxjhe5oqcwal3xfy7fa