Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

5A, NW12/2003G118

Structural analysis of reaction intermediates and mutants of heme oxygenase
Masaki UNNO*, Toshitaka MATSUI, Masao IKEDA-SAITOIMRAM, Tohoku University, Sendai, Miyagi 980-8577, Japan
Introduction
Heme oxygenase (HO) catalyzes oxidative conversionof iron protoporphyrin IX (heme hereafter) to biliverdin
IX, iron, and CO. The enzyme is not a heme protein byitself and uses heme as both the prosthetic group andsubstrate. In catalytic cycle of mammalian enzymes, HO
first binds one equivalent of heme to form a ferric hemeHO complex. The first electron supplied by NADPH
through NADPH-cytochrome P450 reductase converts theheme iron to the ferrous state. Then O2 binds to reduced
penta-coordinate heme to form a meta-stable oxycomplex. One-electron reduction of the oxy form
generates a ferric hydroperoxo species, which selfhydroxylates the α-meso carbon of the porphyrin ring.
Ferric α-meso-hydroxyheme is then converted tobiliverdin by multiple oxidoreductive steps involving averdoheme intermediate.
Mutational studies on polar residues in the distal helixof mammalian heme oxygenase (HO-1) have uncovered a
critical residue, Asp140, whose replacement have lead todominant formation of the inactive ferryl heme. Thedistal Asp is located far from the heme iron (~8 Å) but
forms a H-bond network to an iron-bound water ligand
through intervening water molecule(s). Since this distal
Asp mutation is not expected to disrupt the distal helix
structure, its has been proposed that Asp140, its
carboxylate moiety in particular, is crucial in activation ofFe-OOH through the intervening waters. The distal Asp
is highly conserved in most mammalian, plant andbacterial HOs, whereas HemO from Neisseria
meningitidis and PigA from Pseudomonas aeruginosalack this distal Asp. Absence of carboxyl residue in theirdistal heme pockets has been confirmed in the crystal
structures of HemO and PigA. Despite the absence of thedistal Asp residue, both HemO and PigA are considered
to utilize Fe-OOH in hydroxylation of the heme mesocarbon to form meso-hydroxyheme as mammalian HO.
These findings have raised questions about the generalimportance of the distal Asp carboxylate proposed for
mammalian HO-1, and thus, its reassessment is needed toaccurately delineate how Fe-OOH hydroxylates the heme
meso-carbon.
Result and discussion
To confirm the mutation-induced changes in the waternetwork, crystal structures of the Asp136 HmuO (HOfrom Cryneabacterium diphteriae) mutants have been
examined. The D136N and D136A mutants as well as thewild type crystallized in a space group P21 and theirasymmetric units consisted of three protein molecules.

Only the D136E mutant gave C2 crystals containing twoprotein molecules in the asymmetric unit. Overallstructures and positions of heme group are almost
identical among the wild type and Asp136 mutants (r.m.s.
deviations of Cα are within 0.3 Å), and the major
structural changes are limited to distal heme pockets.
The longer Glu side chain in D136E HmuO directed tothe heme center to diminish its direct interaction withTyr53 and Arg132 present in the wild type HmuO
structure. Arg132 in D136E HmuO rotated away fromheme and forms a H-bond with a new water molecule(W7) without considerable movement of Tyr53. This
structural change of Arg132 should have little effect on
the HO catalysis because rat HO-1 mutants at thecorresponding Arg (Arg136) catalyzed the heme
degradation properly. The water molecules in D136E
were clearly seen as in the wild type except for
replacement of W2 by the carboxylate group of Glu136.
The W1 water in the mutant is further from W0, 4.2 Å,compared to that in the wild type, and now connected
through another water molecule.
To our surprise, the side chain of Asn136 in D136N
HmuO flipped away from the heme center and formed anew water bridged H-bond with Tyr53. This “open”
conformation of Asn136 may be partly due toincorporation of a few iodide ions, a component of thecrystallization solution, in the distal pocket of D136NHmuO; however, wild type and D136E HmuO did notinclude the large ions at the similar condition. Thus, it islikely that Asn136 intrinsically prefers, to a certain extent,
the “open” conformation. Any attempts to remove thelarge ions were failed to prevent us from discussing thewater network for this mutant.
D136A HmuO had similar protein conformation to
those of D136E and D136N HmuO including the positionof Tyr53 and the moved Arg132 side chain (Fig. 6d).
Nevertheless, the water network in D136A HmuO is
totally different from those in wild type and D136E
HmuO. Electron density of W1 was no longer observedin molecule A of D136A HmuO, while W2 and W3
remained at their original positions. The molecule B ofD136A HmuO also lacks W1, whereas the nearby water
was found at 3.1 Å from W0 in the C molecule. Partial
disappearance of W1 is consistent with the small shift ofν(N–O), high reactivity with H2O2 and the ~50 %formation of ferryl heme in the Ala mutant. These results
support significance of the nearby water in activating Fe–
OOH to promote meso-hydroxylation by HmuO.
References[1] T. Matsui, et al., J. Biol. Chem. 280, 2981 (2005).
*unno19@tagen.tohoku.ac.jp

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