Flavohemoglobin: Structure and reactivity


  • Alessandra Bonamore,

    1. Department of Biochemical Sciences, University of Roma ‘La Sapienza’, Piazzale Aldo Moro 5, Roma, Italy
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  • Alberto Boffi

    Corresponding author
    1. Department of Biochemical Sciences, University of Roma ‘La Sapienza’, Piazzale Aldo Moro 5, Roma, Italy
    • Department of Biochemical Sciences, University of Roma ‘La Sapienza’, Piazzale Aldo Moro 5, I-00185 Roma, Italy
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    • Fax: +39-06-4440062


Flavohemoglobins (flavoHbs) are made of a globin domain fused with a ferredoxin reductaselike FAD- and NAD-binding modules. These proteins are widely represented among bacteria and yeasts and represent a most challenging research subject in view of their high reactivity both as reductases and as oxidases. The functional annotations of flavoHbs are still controversial, and different physiological roles that are linked to cell responses to oxidative and/or nitrosative stress have been proposed. The flavoHb from Escherichia coli (HMP) 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 NO in the culture medium, and an explicit mechanism has been proposed that involves NO scavenging and its reduction to N2O under anaerobic conditions. In contrast to (or together with) the anaerobic NO-reductase activity, HMP has also been shown to be able to catalyze the oxidation of NO to NO3 (NO-dioxygenase activity) both in vivo and in vitro in the presence of O2 and NADH. HMP has also been shown to be capable of catalyzing the reduction of several alkylhydroperoxide substrates into their corresponding alcohols using NADH as an electron donor. The alkylhydroperoxide reductase activity taken together with the unique lipid-binding properties of HMP suggests that this flavoHb may be involved in the repair of the lipid membrane oxidative damage generated during oxidative/nitrosative stress. © 2007 IUBMB IUBMB Life, 60(1): 19–28, 2008


The common perception of hemoglobins (Hbs) and myoglobins (Mbs) as proteins tailored for the transport and storage of O2 is not adequate to describe the functional properties of globins within all living organisms. In fact, the discovery of globin genes among prokaryotic and eukaryotic microorganisms including bacteria, yeasts, algae, protozoa, and fungi rules out the possibility that the globin genes were originally designed for O2 transport or storage. At present, the number of these “Hb-like” proteins is steadily increasing as novel genomes from microorganisms are sequenced and annotated. Such a massive input of newly discovered Hbs reveals an unsuspected and amazingly wide distribution of globin genes within living organisms that are distant from the phylogenetic point of view. In this framework, within the evolutionary time frame, O2 transport appears as an emergent function, customized from much older physiological roles, whose nature is under active investigation by the scientific community1, 2.


GCS, globin-coupled sensor; flavoHb, flavohemoglobin; HMP, flavoHb from Escherichia coli; FHP, Alcaligenes eutrophus flavoHb; VHB, Vitreoscilla Hb; trHb, truncated hemoglobin; Mb, myoglobin; FNR, fumarate and nitrate reduction; CFA, cyclopropanated fatty acids; UFA, unsaturated fatty acids; GC, gas chromatography; ESI, electrospray mass spectrometry.

The Globin Superfamily

All globins are structurally related to vertebrate Hbs and Mbs in that, the heme is always covalently linked to the polypeptide chain through a proximal histidine residue (His(F8)) besides the conservation of the typical globin fold, and a Phe residue (Phe(CD1)) appears to be necessary to keep the heme in the correct orientation within the pocket. However, apart from His(F8) and Phe(CD1), a wide array of different aminoacidic residues can be accommodated within the heme pocket. In fact, several topological positions that are important for the interaction of amino acid side chains with the iron-bound ligand in the distal pocket are not conserved among globins. A typical example concerns the topological position E7, which in vertebrate Hbs and Mbs is a His residue but is changed to Gln in several species. When exploring the structural features of the (randomly distributed) invertebrate globins, the variability of the amino acid substitutions within the distal heme site becomes impressive, such that in some nematode Hbs none of the distal heme pocket residues are conserved with respect to the amino acid residues present in higher vertebrate. Climbing the phylogenetic tree up to unicellular microorganisms, an explosion of different globins is observed such that a widening of the parameters defining the globin family is required. In fact, in many microorganisms, including bacteria, yeasts, algae, protozoa, and fungi, the classical globin fold is not fully conserved. Microbial globins include more than a globin family, namely truncated hemoglobins (trHb), globin-coupled sensors (GCSs), and flavohemoglobins (flavoHbs). GCSs include a number of chimeric proteins in which the globin domain is fused with a transducer domain involved in aerotaxis or gene expression regulation3. Interestingly, the globin domain of GCSs and, in particular, the heme-containing active site is structurally analogous to the globin domain of flavoHbs4. Thus, flavoHbs can be considered as a distinct class of chimeric proteins in which the nonglobin module is a typical flavodoxin reductaselike domain5. On the other hand, trHbs share little sequence and structure similarity to O2 sensor domains in that they lack two of the six helices typical of the globin fold and exhibit a wide array of possible amino acid substitutions within the active site6. Thus, trHbs have been assigned to a distinct group within the globin superfamily.

The Flavohemoglobin Family

FlavoHbs are widespread within prokaryotes and eukaryotes, although their distribution among different species is apparently hazardous and does not follow a discernable pattern. So far, enterobacteriacae have one flavoHb gene, while actinobacteriacae and streptomyces often possess more than one flavoHb gene, and some unicellular eukaryotes (Candida albicans) share up to five different genes codifying for flavoHbs. The steadily growing input from novel genomes provides a continuous challenge for the phylogenetic classification of flavoHbs such that the yearly upgrade of genomic databases includes at least a handful of novel proteins from different organisms.

The flavoHb family is formed by a very homogeneous group of proteins that share highly conserved active sites in both the heme- and flavin-binding domains. The conserved amino acids within the heme domain include the residues lining the heme pocket on both the proximal and distal sites, thus indicating that there must be a strong region and stereochemical requirement for ligand binding and/or for gaseous ligand diffusion. In parallel, also the amino acid residues responsible for flavin binding are strictly conserved and conform to the typical architecture of flavodoxin-reductase proteins, indicating clearly that the flavin moiety serves as an electron-transfer module from the NADH to the heme. Sequence alignments carried out on separate domains rapidly diverge towards the globin family on one side and flavodoxin or ferredoxin-reductase family on the other, thus suggesting that the flavoHbs have originated from the fusion of a protoglobin ancestor and a flavin-binding domain.

Escherichia coli Flavohemoglobin

The flavoHb from E. coli (HMP) is the protein that are first discovered of the flavoHbs' family. Since its identification in 19917, HMP has been the object of a large number of investigations aimed at unveiling its physiological role and structural properties.

HMP is encoded by a 1191-bp DNA fragment located at 2683.90 kb in the E. coli chromosome. Its promoter overlaps that of the adjacent gly A gene, which is transcribed in the opposite direction. HMP expression is regulated by the FeS protein FNR (fumarate and nitrate reduction), which predominantly functions as a positive transcription factor, though its role of repressor is also important. FNR recognizes a consensus sequence and binds the DNA in response to exogenous signals such as anoxia, redox state, oxidative, and nitrosative stress8. Inspection of the DNA upstream of the hmp gene reveals two potential FNR-binding sites9. One site, 5′-TTGAG-N4-ATCAA-3′, is centered 34 bp upstream of the hmp translation start codon and closely resembles the consensus sequence, 5′-TTGAT-N4-ATCAA-3′; its position is consistent with an inhibiting function. A second potential FNR site, 5′-TTGAC-N4-AGGAA-3′, is centered 222 bp upstream of the hmp translation start codon but has a weaker resemblance to the consensus FNR-binding sequence.

HMP expression appears to be also regulated by Met R. One gene activated by Met R with homocysteine as cofactor is gly A, whose promoter overlaps that of hmp. It has been demonstrated that elevated homocysteine levels in E. coli decrease HMP expression10. Furthermore, induction of HMP expression was observed in cells entering the stationary growth phase. This induction is mediated by the stationary phase-specific sigma subunit (σS) of RNA polymerase11.

Later on, based on the impetus provided by the active and successful research on the biological effects of NO and on multiple experimental evidences of an interplay between the NO catabolism and Hb chemistry, it was proposed that HMP might act in connection with novel NO-related pathways in bacteria12–14. Other functional hypotheses have been suggested that entails the participation of HMP to a general oxidative/nitrosative stress response as a lipid-specific alkylhydroperoxide reductase15.


The structure of flavoHbs represents a paradigmatic example among multidomain proteins in which two modules with different functions are fused together to create a protein endowed with entirely novel functional properties. The first X-ray structure of flavoHb (Alcaligenes eutrophus flavoHb, FHP), obtained in 1995 by Ermler and coworkers16, had shown that these flavoHbs are made of a C-terminal NAD- and FAD-binding domain, a member of the FdR-like family, and an N-terminal globin domain (Fig. 1). Surprisingly, inspection of the structure of the globin domain active site in FHP revealed that the distal heme pocket was occupied by a phospholipid molecule. The presence of this bulky ligand was apparently unrelated to possible functional roles of FHP and rendered the understanding of the geometry of the heme active site particularly difficult. The crystallization of the highly homologous HMP in its lipid-free form finally disclosed the architecture of the active site17. The overall structure of ferric unliganded HMP, obtained at 1.6-Å resolution, brings about strong analogies with the structure of FHP, although HMP crystals have been obtained on the fully oxidized protein (i.e., heme iron in the ferric state and oxidized flavin), whereas the structure of FHP was determined on the fully reduced protein (i.e., heme iron in the ferrous state and reduced flavin).

Figure 1.

Overall three-dimensional structure of HMP. The heart-shaped structure is positioned with the flavin-binding domain at the upper apex (cyan), the globin domain on the lower right side (red), and the NAD-binding domain on the lower left side (green). The ribbon diagram was depicted using the program MOLSCRIPT (http://www.avatar.se).

The overall fold of flavoHbs (Fig. 1) consists of a heart-shaped structure in which three different domains, namely the C-terminal NAD-binding domain, the FAD-binding domain, and the N-terminal globin domain are clearly distinguished.

The Globin Domain

The architecture of the globin domain corresponds to a classical globin fold composed of six helices (from A to H) with an unusually long H-helix; the D-helix is substituted by a large loop region within the segment Asn44(CD2)-Asp52(E6). Apart from the invariant residues Phe(CD1) and His(F8), considered as the hallmark of the globin structure, the heme pocket geometry shows little resemblance to that observed in vertebrate Hbs and Mbs. Inspection of the distal pocket architecture reveals that the distal position, closest to the iron atom, is occupied by the Leu57(E11) isopropyl side chain with the CG atom at 3.6-Å distance from the iron atom along the normal to the heme plane (Fig. 2). The Leu57(E11) side chain, together with the Phe43(CD1) ring, the Ile61(E15), and Val98(G8) side chains and the Gln53(E7) backbone segment completely fill the first distal shell (Fig. 2). In contrast, Tyr29(B10) and Gln53(E7) side chains, previously proposed as the major candidates for iron-ligand distal stabilization16, are shifted from the heme at a distance of more than 5.0 Å. The Tyr29(B10) phenol ring is confined to the second layer of distal pocket residues with the phenol hydroxyl pointing towards the isobutyl chain of Leu57(E11)17.

Figure 2.

Structural details of the heme-binding site in ferric-unliganded HMP. The heme group is shown together with a selection of amino acid residues located within 5 Å distance from the macrocycle atoms. The picture was obtained with the program ViewerLite (http://www.accelrys.com/products/).

The architecture of the heme pocket in the proximal region is characterized by the presence of the canonical proximal His residue (His85(F8)) whose ND1 atom coordinates the ferric heme iron at 2.01-Å distance. However, at variance with vertebrate Hbs, the proximal His residue is involved in a hydrogen-bonding network comprising His85(F8), Tyr95(G5), and Glu135(H23) (Fig. 2) that imparts a rigid orientation to the imidazole ring of His85(F8) with respect to the heme plane. In particular, the OE2 atom of Glu135(H23) is involved in a hydrogen-bonding interaction at 2.9 Å from the ND1 nitrogen of the proximal His85(F8). In turn, the OE2 atom of Glu135(H23) is anchored to the phenol hydroxyl of Tyr95(G5) by a hydrogen bond (2.7 Å).

The structural overlay of the Cα skeleton of HMP and FHP globin domains indicates that a major difference between the two flavoHbs pertains to the positioning of the E-helix that is shifted far from the heme plane in the latter protein and is determined by the accomodation of a bulky phospholipid molecule that alters the positioning of the relevant distal pocket residues18. In particular, in FHP, the cyclopropane ring of a 9,10-methylen-hexadecanoic chain of a dyacylglycerol-phosphatidic acid (a common fatty acid component within the bacterial membranes) sits on the top of the iron atom apparently displacing the relevant distal site residues. In fact, in FHP, Leu57(E11) accompanies the lipid-induced E-helix movement and is displaced by 4.6 Å away from the heme pocket with respect to its positioning in HMP. Interestingly, HMP is also capable of binding membrane lipids, although with lower affinity with respect to FHP18, 19, and VHB has also been recently reported to be able to interact with the bacterial inner membrane20, 21. Taken together, these observations suggest that lipid binding is not a distinctive feature of FHP but may represent a common property of the bacterial Hb family whose functional role is still to be defined.

The FAD-binding Domain

The structures of the FAD- and NAD-binding modules in HMP are superimposable to those observed in FHP. The former consists of a six-stranded antiparallel β-barrel with Greek key topology capped by a helix at the bottom and an irregular peptide at the top. The latter is built up of a five-stranded parallel β-sheet flanked by two helices on one side and by one helix and an irregular structural element on the other. The FAD- and NAD-binding domains are fused to build the so-called oxidoreductase module, which structurally belongs to the ferredoxin-reductase family.

Nevertheless, two major differences between HMP and FHP need to be considered further. First, as shown in Fig. 3, the relative orientations of the FAD- and NAD-binding domains in the two proteins differ. In particular, the entire NAD-binding domain in HMP is rotated clockwise with respect to the relative orientation observed in FHP, thus leading to a considerably larger interdomain cleft in HMP. The second point concerns the structural arrangement of the isoalloxazine-binding motif. The si-side geometry is strikingly similar in the two proteins in that the segments 205–207 (207–209 in FHP) and the Tyr188 (190 in FHP) exhibit identical orientations with respect to the isoalloxazine plane (see Fig. 3). Other conserved contacts (not shown) to the flavin moiety involve Val269 in HMP (276 in FHP) and Thr272 (279 in FHP). In contrast, the re-side that faces the C-terminus loop region of the NAD-binding domain is quite different in the two proteins. In fact, in HMP, the aromatic ring of the conserved Phe390 (396 in FHP) is packed against the isoalloxazine central ring, whereas in FHP, it protrudes towards the interdomain cleft. Considering the high sequence similarity within the FAD- and NAD-binding domains of the two flavoHbs, both the rotation of the NAD-domain and the differences in the re-side of the isoalloxazine-binding motif may be suggestive of a conformational change linked to the binding of a phospholipid molecule. Thus, it may be envisaged that the lipid-free species is stabilized by a stacking interaction of the isoalloxazine ring with Phe390 (in HMP), whereas, upon lipid binding (i.e., in FHP), the whole C-terminus loop is shifted and rotated toward the ribitol moiety with concomitant loss of the stacking interaction16, 17. Despite these significant structural rearrangements, the relative positioning of the isoalloxazine moiety and the heme plane is superimposable in the two flavoHbs, and only a modest decrease in the closest distance between the two cofactors is observed (the distance between the C8AF atom of the isoalloxazine and the OA1 atom of the heme propionate is 6.3 Å in FHP and 5.9 Å in HMP).

Figure 3.

Structural details of the FAD-binding site in ferric-unliganded HMP (right) and ferrous, lipid-bound FHP (left). The isoalloxazine three-membered ring (embedded in a transparent van der Waals surface) is shown together with a selection of amino acid residues located within 5 Å distance from the macrocycle atoms in the re-side (top) and in the si-side (bottom). Pictures were obtained with the program ViewerLite (http://www.accelrys.com/products/).

Structure-based Sequence Alignment

The clarification of the structural properties of HMP with respect to FHP and the understanding of the architecture of the active sites of these proteins allowed a rational evaluation of the structure-based alignments of the whole flavoHbs family. Sequence alignments were carried out on Gram-negative bacteria (enterobacteriaceae, vibrionaceae, pseudomonaceae, and rhizobiaceae) as well as on Gram-positive bacteria (bacillaceae and micrococcaceae) and even unicellular eukaryotes. The flavoHb family is formed by a homogeneous group of proteins that share highly conserved active sites in both the heme- and flavin-binding domains. A multiple sequence alignment of about 115 proteins from different bacterial and eukaryotic microorganisms shows that the residues surrounding the heme cofactor in HMP are conserved in all flavoHbs analyzed to date. The residues contouring the flavin-binding cavity are also conserved (Fig. 4).

Figure 4.

Conserved residues within the flavohemoglobin family. HMP heme domain (upper panel) and flavin domain (lower panel) are shown. The amino acids represent residues conserved among 115 flavoHbs identified so far in genomic databases (BLAST search; http://expasy.org/tools/blast/) and CLUSTAL W analysis (http://www.ebi.ac.uk/clustalw/). Pictures were obtained with the program ViewerLite (http://www.accelrys.com/products/).

Invariant residues close to the heme cofactor correspond to Phe28(B9), Tyr29(B10), Phe43(CD1), Gln53(E7), Leu57(E11), Asn44(CD2), Val98(G4), Leu102(G8), and Tyr124(H11) on the heme distal side and to His85(F8), Glu135(H22), Tyr95(G1), and Lys84(F15) on the heme proximal side of HMP (see Fig. 4). Phe43(CD1) is absolutely conserved in all species, and its aromatic side chain binds nearly parallel to the heme plane. The proximal His85(F8) residue is also invariant in all known globin sequences, even if its environment changes remarkably. Within the flavoHb family, His85(F8) is hydrogen bonded to the conserved Glu135(H22) and Tyr95(G1). The distal Tyr29(B10) and Gln53(E7) are supposed to be involved in the stabilization of O2 bound to the heme iron. Finally, Phe28(B9), Leu57(E11), Val98(G4), and Tyr124(H11) seem to be involved in phospholipid binding.

The conserved amino acids of the flavin domains correspond to the following residues: Phe290, Gly270, Glu388, Thr272, Pro273, Gly186, Gln187, Tyr188, and Ser232. These residues occupy topological positions that are highly distinctive of ferredoxin (or flavodoxin) reductases, a subfamily of flavin-binding proteins that is highly specialized in electron transfer from the NADH cofactor to the flavin moiety and to a high-redox potential electron acceptor, typically a ferredoxin. Consistently, the conserved residues appear to contour and shape the flavin-binding cavity, thus reducing the solvent access and providing the structural basis for the interaction with NADH as a substrate.


Lipid-binding properties

The interaction of bacterial Hbs with membranes has been first proposed for Vitreoscilla Hb (VHB), which has been demonstrated to bind the inner membrane and act as a facilitator of O2 transfer by placing it closest to the respiratory chain. FHP is also able to bind membrane phospholipids16, 18. The X-ray structure of HMP not only revealed a high degree of similarity with both VHB and FHP but also showed that the amino acid residues involved in the binding of the lipid molecule are strictly conserved.

Based on this, the fatty acid and phospholipid-binding properties of ferric HMP in solution were investigated by spectroscopic, thermodynamic, and kinetic methods19. In simple UV–vis absorption experiments, it was demonstrated that ferric HMP is capable of interacting with bacterial lipid membranes and is also able to recognize and bind specifically unsaturated fatty acids (UFA) and cyclopropanated fatty acids (CFA). Thus, the spectral profile of lipid-bound HMP is diagnostic to the formation of a ferric hexacoordinate species in which the sixth coordination position is occupied by a component of the fatty acid chain. It has been proposed that the double bond or cyclopropane ring recognition by flavoHb involves a weak but direct-bonding interaction to the heme-iron atom. Interestingly, the ferric ligand-free HMP (1.6-Å resolution) displays similar structural features within the iron first coordination shell with the CG carbon atom of Leu57(E11) at 3.4-Å distance from the iron atom. In agreement with the crystallographic data, optical absorption (both in solution and in crystals) and resonance Raman spectroscopy point out unambiguously that the lipid-free ferric derivative is a pentacoordinate species, and the Leu57(E11) residue does not establish a bonding interaction with the ferric iron. In contrast, the formation of a hexacoordinate derivative is clearly demonstrated upon lipid binding to the ferric HMP protein by the low frequency shift of the core size marker bands22. The appearance of a hexacoordinate high spin species is accompanied by an unusually intense band at 319 cm−1 attributed to a γ6 heme out-of-plane vibrational mode. The conversion from a pentacoordinate to a hexacoordinate species is consistent with the formation of an axial bonding interaction between the ferric heme iron and the lipid moiety. Thus, on the basis of the structural data of Ollesch and coworkers18, we proposed an explicit model for UFA or CFA binding to HMP in which the negatively charged part of the lipid moiety is hosted in an anion-binding cavity located next to the heme pocket, and the cyclopropane ring or the double bond is directly coordinated to the iron atom. To single out the nature of such an unusual iron-ligand-bonding interaction, EXAFS measurements have been carried out in parallel to the ligand-free and lipid-bound protein. Quantitative analysis of the EXAFS data indicates that a bonding interaction does occur between the ferric heme iron and a couple of atoms located at 2.7 Å from the metal22.

A complete set of kinetic and thermodynamic parameters for fatty acid binding to ferric HMP has been obtained for linoleic acid in the presence of ethanol (20%) as a cosolvent. The affinity-binding constant, estimated from ligand binding and release data is about 4 × 107 M−1 (or Kd = 25 nM), a figure that is similar to that of true-type fatty acid-binding proteins23. The identification of an anion-binding site (fully compatible with a phosphate-binding site) in a protein loop above the heme pocket allows one to hypothesize a recognition of the membrane through an interaction of the loop with the phosphate moiety of the phospolipid molecule. Thus, HMP has been proposed to act at the interface between the cytosol and the bacterial inner membrane and to be permanently saturated with a phospholipid esterified with either a UFA or a CFA molecule.

Ferrous Heme-iron Ligand-binding Properties

Ligand-binding kinetic properties of ferrous HMP have been investigated by measuring the second-order binding rates of CO and the first-order kinetics of O2 release on both the lipid-free protein and the HMP saturated with different lipids or fatty acids. Lipid-free HMP yielded biphasic second-order CO-binding kinetics [k1 = 1.8 × 107 M−1 s−1 (68%) and k2 = 1.2 × 106 M−1 s−1 (32%)]24. In contrast, experiments carried out on HMP saturated with E. coli lipid extracts yielded perfectly monophasic CO ligand rebinding time courses after photolysis with a second-order rate of 5.1 × 106 M−1 s−119. In both lipid-free and lipid-saturated protein, no geminate recombination processes were detected as demonstrated by the flatness of the nanosecond time recording.

Kinetics of O2 release, as monitored in O2 pulse experiments, is likewise biphasic in lipid-free HMP with first-order rates (koff) of 2.2 s−1 (44%) and 0.11 s−1 (56%). In contrast, O2 release from the total lipid extracts saturated protein is accounted for by a single exponential decay (koff = 0.16 s−1 at 20°C). Direct O2-binding kinetics has not been reported.

Given the significant effect on the kinetic parameters of CO and O2 binding/release, it must be envisaged that the lipid acyl chain is present in the heme pocket, thus impairing CO binding and favoring the O2 release processes. In turn, the lipid-free protein must be endowed with a conformational equilibrium between two species to explain the heterogeneity observed in CO binding and O2 release. These hypotheses (that are not mutually exclusive) may be reconciled with the previous findings based on resonance Raman25 and infrared spectroscopic26 investigations (carried out on the lipid-free protein), which envisaged the presence of two distinct conformers in the CO-ligated species. The presence of two separate CO-stretching frequencies had been attributed to the presence of an “open” and “closed” conformers25. In the “closed” conformer, it has been suggested that the heme-iron-bound CO is stabilized by the Tyr29(B10) phenol hydroxyl, whereas it is apparently free of constraint in the “open” conformation25, 26.

Ferric Heme-iron Ligand-binding Properties

The understanding of ligand-binding properties in ferric Hbs is a key step to the comprehension of the structural factors governing ligand entry or escape to/from the active site. In flavoHbs, these properties are at present less investigated. As an example, the broad, biphasic equilibrium titration profiles for ligand (cyanide, azide, and imidazole) binding to ferric lipid-free HMP and VHB, are most unusual. Ligand binding to VHB was initially interpreted as an anticooperative behavior due to the apparently dimeric nature of the protein27. Subsequently, the presence of a dimeric species in VHB has been challenged in an ultracentrifugation investigation, thus leaving the biphasic equilibrium ligand-binding properties unexplained28.

A tentative reaction scheme (Fig. 5) that takes into account the observed ligand-binding profiles has been unravelled on the basis of a set of equilibrium and kinetic measurements carried out on both lipid-bound and lipid-free ferric HMP19. According to Scheme 1, the lipid is weakly coordinated to the heme iron and is strongly bound to the nonheme site. Imidazole binding to the heme iron occurs upon displacement (not rate limiting, at least in the case of imidazole) of the weak iron-lipid bond and is entirely accounted for by a typical single-site ligand-binding process. The nonheme site must possess a very high affinity for the polar lipid head such that it cannot be displaced by heme-iron ligands.

Figure 5.

Reaction schemes for ligand binding to ferric HMP. Ferric HMP has been inferred to possess a heme-iron ligand-binding site (−P) and a nonheme-binding site (P∼). Both sites are involved in the recognition of lipid (UFA or CFA) substrates (L). The interaction of the lipid with the heme site is weak and can be displaced by heme-iron ligands (x), as depicted in Scheme 1. Complete removal of the bound lipid renders the nonheme site available for ligand interaction as outlined in Scheme 2. The ligand binds to the heme iron and is accompanied by a fast ligand exchange (k3 and k−3) between the heme- and the nonheme-binding sites. Full saturation of both sites implies a conformational transition from P to P* species.

In the lipid-free protein, the set of imidazole-binding kinetics and equilibrium measurements suggested that the ligand might be able to occupy a protein site or cavity next to the heme that allows for rapid exchange with the heme iron. It is envisaged that the incoming ligand is coordinated by the heme iron (k1′ > k2′) and then rapidly partitioned (k3′ ≈ k3 > 1000 s−1) between the metal and the spectroscopically silent nonheme site (Scheme 2).

This scheme is in agreement with the presence of the two sites previously postulated for UFA recognition, that is, the heme-iron site that is capable of coordinating the acyl chain double bond (or cyclopropane ring) and a polar cavity that is necessary for harboring the polar head of the lipid. Thus, heme-iron ligands might be hosted by the polar site in competition with the heme iron in the lipid-free HMP. The main question that arises from the proposed mechanism concerns the nature of the nonheme-binding site and its unusually high affinity for the typical ferric heme-iron ligands.


NO Dioxygenase Activity

HMP has been demonstrated to play a role in the framework of bacterial resistance to nitrosative stress. So far, HMP expression responds to the presence of NO in the culture medium12, 29–31, and an explicit mechanism has been proposed that involves FNR-mediated NO-induced expression of the flavoHb9, 32, 33. HMP has been shown to be able to scavenge and reduce NO to N2O under anaerobic conditions34. 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 NO3 (NO-dioxygenase activity) both in vivo and in vitro in the presence of O2 and NADH14. Alternatively, a NO denitrosylase function has been proposed in which, at low O2 tensions, HMP turns over in the ferric state with the intermediacy of an iron-bound nitroxyl anion that is subsequently transformed into NO3 in the presence of O235. It remains to be established which of these diverse enzymatic activities might correspond to a physiologically relevant process36, 37.

HMP is endowed with a robust NO-dioxygenase activity24. Reduction by NADH occurs in two steps. NADH reduces bound FAD with a rate constant of ∼15 μM−1 s−1, and heme iron is reduced by FADH2 with a rate constant of 150 s−1. O2 binds tightly to reduced HMP, with association and dissociation rate constants equal to 38 μM−1 s−1 and 0.44 s−1, respectively, and oxygenated HMP dioxygenates NO to form NO3. NO also binds reversibly to reduced HMP in competition with O2, dissociates slowly and inhibits NO-dioxygenase activity at the [NO]/[O2] ratio of 1:100. At 37 °C, Vmax/Km(NO) is 2400 μM−1 s−1, demonstrating that the enzyme is extremely efficient at converting toxic NO into NO3 under physiological conditions.

The NO dioxygenation reaction rate constant is 40–70 times greater than the values of 35 and 60 μM−1 s−1 for mammalian MbO2 and HbO2, respectively, and is only approximately two times lower than the diffusion-limited rate constant for the reaction of NO with free O224. The high in vivo (60 μM) and in vitro (80–100 μM) values for the Km(O2) at 37°C indicate that O2 concentration will limit NO-dioxygenase activity under physiological conditions. The strong dependence of NO-dioxygenase activity on [O2] may explain the benefit for hmp upregulation in microbes in response to lower O2 availability. Higher levels of HMP will be required to compensate for decreased NO-dioxygenase activity at a low [O2]. In turn, low NO-reductase activity (0.013–0.24 NO heme−1 s−1) has been reported11, 12 that is more than 1000-fold lower than the NO-dioxygenase activity.

Alkylhydroperoxide Reductase Activity

HMP is able to reduce O2 to O238 as well as H2O2 to H2O15 at the expense of NADH-reducing equivalents and under low O2 content. The H2O2-reduction activity differs from the classical catalase activity (H2O2 disproportionation to yield H2O and O2) in that it entails H2O2 reduction to H2O without O2 production. This enzymatic activity has been formerly defined as peroxide reductase activity and is typical of thiol-based proteins peroxiredoxins39, 40. In turn, the peroxiredoxin family comprises enzymes that are also capable of reducing alkyl hydroperoxides and the alkyl hydroperoxide reductases41.

Ferrous HMP has been demonstrated to possess a genuine alkyl hydroperoxide reductase activity in anaerobiosis, thus suggesting that HMP itself and possibly other members of the Hb-like protein family can be involved in the reduction of lipid-membrane hydroperoxides. The hydroperoxide reductase activity of HMP was screened by using H2O2, tert-butyl hydroperoxide, cumyl hydroperoxide, and linoleic acid hydroperoxide as substrates. The reaction products, analyzed by HPLC, GC, or ESI mass spectrometry, revealed transformation of the alkyl hydroperoxide species into their corresponding alcohols.

The highest activity was observed for cumyl and linoleic acid hydroperoxides, whereas the reduction of tert-butyl hydroperoxide and H2O2 was two and threefold slower, respectively (Table 2). It should be pointed out that the reaction with linoleic acid hydroperoxide is strongly solvent dependent. Data obtained in the presence of 0.1% Triton X-100 were fully reproducible and displayed nearly zero-order kinetics. In contrast, experiments carried out in 20% ethanol/water mixture (in which the substrate is soluble up to 2–3 mM concentration at 25°C) exhibited a slightly slower rate and displayed nearly exponential time courses.1

Table 1. Steady-state parameters for the NO-dioxygenase activity of HMPa
Steady-state parameterbSubstrate
  • a

    Data were obtained at pH 7.0 (0.1 M phosphate buffer containing 250 mM NADH and 1.0 mM EDTA) and 25°C.

  • b

    Vmax and Km values were determined from initial velocities. Km values for NADH, NO, and O2 were calculated from the transient state parameters from24.

  • c

    The substrate concentrations exceeded largely the Km values24.

Vmax = 94 s−1NADH, NO, O2c
Km = 3.2 μMNADH
Km = 0.11 μMNO
Km = 27 μMbO2
Table 2. Steady-state parameters for the alkylhydroperoxide reductase activity of HMPa
SubstrateVmax (nmol min−1 mg−1)Km (μM)
  • a

    For comparison with data from other authors, activities can be redimensioned ‘per mol’ using the molecular mass of 43,000 g mol−1 for HMP. Data were obtained at pH 7.0 (0.1 M phosphate buffer containing 250 μM NADH and 1.0 mM EDTA) and 25°C19.

  • b

    0.1% Triton X-100.

  • c

    20% ethanol/water mixture.

Tert-butyl hydroperoxide112576
Cumyl hydroperoxide256455
Linoleic acid hydroperoxide1876b26b

Competitive inhibition by CO was also observed for cumyl hydroperoxide, thus demonstrating that the hydroperoxide reductase activity entails hydroperoxide binding to the heme iron. From the mechanistic point of view, the alkylhydroperoxide reductase activity of HMP can be rationalized as follows. HMP is capable of recognizing the alkyl hydroperoxide moiety due to the highly hydrophobic distal heme pocket and, in the case of peroxidized phospholipids containing UFA, has been inferred to be able to adjust the hydrocarbon chain kink in correspondence to the cis double bond above the heme iron. Thereafter, binding of the hydroperoxide to the ferrous heme iron occurs with the concomitant two electron reduction and cleavage of the dioxygen bond and consequent formation of a peroxidaselike compound II and an alkyl alcohol. It is important to note that this first step does not imply electron transfer from the flavin to the heme iron but rather a direct two-electron iron oxidation. The fate of the ferryl-oxo compound, thus generated, is less obvious. Reduction of the ferryl-oxo compound to H2O and ferric heme with concomitant flavin oxidation and release of the hydroxylphospholipid to the membrane may be hypothesized. Most intriguingly, the active form of the protein, capable of binding a new molecule of substrate, is the ferrous derivative and not the ferric one. In fact, at variance with classical peroxidases, no alkyl hydroperoxide and/or H2O2 binding to the heme iron can be detected when the heme iron is in the ferric state.

At present, a direct, in vivo, determination of the HMP-dependent alkyl hydroperoxide reductase activity still needs to be explored. Nevertheless, a number of experimental observations on HMP expression in response to oxidative stress conditions provide convincing evidence for a genuine physiological role of the protein in the repair of oxidative damage. In particular, it was observed that the HMP expression, controlled by the FNR transcription factor, is enhanced under conditions of prolonged oxidative stress, such as those imposed by administrating paraquat and other agents generating oxygen-reactive species to the culture medium42. This finding is consistent with HMP being transcribed in a later phase after the oxidative pulse. In fact, whereas the fast response to oxidative stress is governed by enzymes that are directly under the control of the transcription factor OxyR39, the late response to oxidative stress involves a web of interactions that ultimately lead to HMP transcription42. Thus, enzymes involved in H2O2 scavenging (AhpCF and KatG/E gene products) or DNA protection (Dps gene product) are rendered available as a first barrier against peroxidation, whereas HMP and possibly other substrate-specific enzymes may play a role in the repair of diverse peroxidized species. In this framework, the link between the reported NO-induced (FNR mediated) expression of HMP32 and oxidative stress may reflect another facet of the complex and intimately correlated responses of the bacterial cell to NO and the oxygen-reactive species. NO induction of enzymes involved in the oxidative stress response is in fact well documented43, and explicit mechanisms that envisage an increase of the alkyl hydroperoxide reductase activity in the presence of NO have been proposed44. In particular, ONOO species, formed at a diffusion-limited rate by the reaction of NO with O2, are known to be a strong promoter of membrane lipid peroxidation45. Accordingly, NO-induced HMP expression, under low O2 tensions, might well allow a direct (FNR mediated) mechanism for lipid hydroperoxide reduction, whereas peroxiredoxins actively scavenge ONOO44.


The overall picture that emerges from more than a decade of biochemical and microbiological investigations on flavoHbs indicates that these proteins are key enzymes in maintaining the cell redox hoomeostasis at the aerobic/anaerobic interface when bacterial cells are exposed to oxidative/nitrosative stress. Specific mechanisms linked to NO detoxification have been demonstrated, although other catalytic options and physiological functions must be taken into account for further studies. In fact, lipid hydroperoxide reductase activity might be part of a specific reaction pathway that leads to the formation of lipid hydroperoxide-derived active compounds whose functions are, as yet, to be established. Thus, bacterial Hbs might be involved in the (possibly NO driven) processing of phospholipids in the framework of a more complex physiological response.


National grants from MIUR (Ministero dell'Università e della Ricerca) FIRB 2003 to A.B. are gratefully acknowledged.