An enzymatic globin from a marine worm

Martin

Journal of Biological Chemistry

et al. 2000

155

166

The flavoHbs 1 belong to a 1.8 billion-year-old family of globin molecules that includes O 2 -binding Hbs and Mbs isolated from animals, plants, fungi, protozoa, bacteria, and worms (1-6). FlavoHbs have a unique two-domain structure containing linked Hb and reductase domains with extensive homology to the mammalian Hbs and metHb reductases (1, 7). Other Hbs appear to be co-expressed with associated metHb reductases (8). An O 2 transport or storage function, like that of the erythrocytic Hbs and muscle Mbs, has been suggested for some microbial and plant (flavo)Hbs (4, 9); however, other functions including the catalysis of oxidations have long seemed more likely (10 -12).Recently, we described an NO dioxygenase (NOD) produced by Escherichia coli that utilizes O 2 and NAD(P)H to convert NO to nitrate (Equation 1) and identified it as a flavoHb (13,14). Subsequent studies have shown that related bacterial and yeast flavoHbs display a similar NOD activity.A role for flavoHbs in NO detoxification is supported by the ability of flavoHbs to protect bacteria against NO or nitroso compounds (13, 14, 16 -18) and by their induction in bacteria exposed to NO, nitrate, nitrite, or nitroso compounds (13, 14, 17, 19 -22). However, the mechanism of NO detoxification, and thus the function of the flavoHbs, is obscured by the possibility of multiple reaction mechanisms involving NO. Other NO detoxifying activities for flavoHbs, including denitrosylation of nitrosothiols (17), NO reduction (17, 23), and NO sequestration (16, 23), have been offered to explain the protection flavoHbs afford to bacteria against NO and nitrosoglutathione. Thus, an understanding of the biological function(s) of the flavoHbs demands a greater knowledge of their various activities.In this report, steady-state, reduction, and ligand binding kinetics of the E. coli NOD (flavoHb) were measured in order to define its function and the mechanism of NO dioxygenation. We also examined the effects of amino acid substitutions at the highly conserved Tyr(B10) position on NOD activity, reduction, and ligand binding kinetics (7,24). Key differences between flavoHb and other Hbs are discussed in light of this specialized but perhaps ancient NO dioxygenation and detoxification function of hemoglobin. MATERIALS AND METHODSCells and Reagents-The flavoHb-deficient E. coli strain RB9060 (25) was generously provided by Alex Ninfa (University of Michigan). Plasmid pAlter containing the E. coli hmp gene was prepared as described previously (13). FAD, NADPH, and bovine hemin were purchased from Sigma. NADH, bovine liver catalase (260,000 units/ml), and deoxyribonuclease were obtained from Roche Molecular Biochemicals. Saturated NO was prepared as described previously (26). Saturated O 2 (1.14 mM) was prepared by vigorously scrubbing a solution of 50 mM potassium phosphate, pH 7.8, containing 0.1 mM EDTA (buffer A) at 25°C and atmospheric pressure with 99.993% O 2 (Praxair, Bethlehem, PA) in a rubber septum-sealed glass vial vented with a syringe needle. Manganese-containing...

Section: Nod Activity and The Function Of Flavohb-it Is Clear From Thmentioning

confidence: 98%

Steady-state and Transient Kinetics of Escherichia coli Nitric-oxide Dioxygenase (Flavohemoglobin)

Martin

Journal of Biological Chemistry

et al. 2000

155

166

Acta Crystallogr D Biol Cryst

“…DHP has been shown to be a homodimer consisting of two identical subunits of $15.5 kDa in the asymmetric unit, each of which contains a heme protoporphyrin IX cofactor and eight -helices (de Serrano et al, 2007;Zhang et al, 1996). These subunits are connected through an interface mainly comprised of salt bridges from acidic and basic amino-acid residues on the protein surface, specifically between Asp72 of one subunit and the side-chain groups of Arg122 and Asn126 of the other subunit (Lebioda et al, 1999;Chen et al, 2009). There is also a hydrophobic interaction between the Val74 residues of the two subunits (LaCount et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

“…To date, X-ray diffraction studies of dehaloperoxidase have been limited to one isoenzyme (DHP A; Chen et al, 2009; de Serrano et al, 2007;LaCount et al, 2000;Zhang et al, 1996;Lebioda et al, 1999). However, two isoforms of dehaloperoxidase, termed DHP A and DHP B, occur in A. ornata and are encoded by two separate genes (dhpA and dhpB; Han et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

“…One intriguing possibility is that the distal histidine His55, which has been observed in distinct 'open' and 'closed' conformations, mediates either H 2 O or O 2 displacement from the heme in the presence of (tri)halophenol substrate, possibly serving as a trigger for peroxidase function (Chen et al, 2009). It has been hypothesized that the 'open' and 'closed' conformations are the consequence of an equilibrium between five-coordinate and six-coordinate (metaquo) forms of DHP at room temperature, respectively (Chen et al, 2009;Lebioda et al, 1999). Specifically, in the open conformation His55 is swung out of the distal pocket and solvent-exposed, with a heme Fe-N His distance of $9 Å , and for this reason is unable to participate in hydrogen-bonding interactions that would stabilize the presence of a sixth axial heme ligand.…”

Section: Introductionmentioning

confidence: 99%

“…Since the seminal work of Lebioda and coworkers revealed a small-molecule-binding pocket in close proximity to the heme active site (Lebioda et al, 1999), several studies have focused on the specifics of substrate localization in DHP (Davis et al, 2009;Smirnova et al, 2008;Nienhaus et al, 2008;Belyea et al, 2005), a feature that is unique to dehaloperoxidase among all known globins. More recently, it has been postulated that both external and internal small-moleculebinding sites may exist, with regulatory implications that may govern how DHP switches between its hemoglobin and peroxidase activities (Chen et al, 2009;Davis et al, 2009;Smirnova et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Structure of dehaloperoxidase B at 1.58 Å resolution and structural characterization of the AB dimer fromAmphitrite ornata

Serrano¹,

D’Antonio²,

Franzen³

et al. 2010

As members of the globin superfamily, dehaloperoxidase (DHP) isoenzymes A and B from the marine annelid Amphitrite ornata possess hemoglobin function, but they also exhibit a biologically relevant peroxidase activity that is capable of converting 2,4,6-trihalophenols to the corresponding 2,6-dihaloquinones in the presence of hydrogen peroxide. Here, a comprehensive structural study of recombinant DHP B, both by itself and cocrystallized with isoenzyme A, using X-ray diffraction is presented. The structure of DHP B refined to 1.58 Å resolution exhibits the same distal histidine (His55) conformational flexibility as that observed in isoenzyme A, as well as additional changes to the distal and proximal hydrogen-bonding networks. Furthermore, preliminary characterization of the DHP AB heterodimer is presented, which exhibits differences in the AB interface that are not observed in the A-only or B-only homodimers. These structural investigations of DHP B provide insights that may relate to the mechanistic details of the H 2 O 2 -dependent oxidative dehalogenation reaction catalyzed by dehaloperoxidase, present a clearer description of the function of specific residues in DHP at the molecular level and lead to a better understanding of the paradigms of globin structure-function relationships.

Structural evidence for stabilization of inhibitor binding by a protein cavity in the dehaloperoxidase‐hemoglobin from Amphitrite ornata

Serrano

Franzen

2011

Biopolymers

A functional role for a protein cavity that stabilizes inhibitor binding has been established based on a comparison of Xe-derivatized and inhibitor-bound X-ray crystal structures in dehaloperoxidase-hemoglobin (DHP A) of Amphitrite ornata. The internal binding affinity of four different inhibitors, 4-fluorophenol, 4-chlorophenol, 4-bromophenol, and 4-iodophenol in the distal pocket has been shown previously to increase proportional to the radius of the para-halogen atom. Inhibition of oxidation of the native substrate, 2,4,6-tribromophenol, has been shown to follow the trend in inhibitor binding strength, because of a two-site competitive inhibition mechanism that involves displacement of the substrate by the inhibitor in a gated mechanism involving the distal histidine of DHP A. In this study, it is shown that the origin of the stronger binding by a larger para-halogen substituent coincides structurally with a Xe-binding cavity (Xe1) characterized structurally by X-ray crystallography. The Xe1 site is surrounded by amino acid resides L100, F21, F24, F35, F60, and V59 in the distal pocket, located 4.8 Å from the heme iron, in a position that is coincident with the para-bromine atom of the inhibitor 4-bromophenol. 4-bromophenol is prevalent in benthic ecosystems where A. ornata resides. A second, less well-defined, binding site in DHP A, labeled as Xe2, is located near the surface of the protein in the vicinity of amino acid residues L62, R69, D79, T82, and L83, which may be related to substrate docking on the surface of DHP A.