ORIGINAL ARTICLE H.-P. Hersleth á B. Dalhus á C.H. GoÈrbitz K.K. Andersson An iron hydroxide moiety in the 1.35 A Ê resolution structure of hydrogen peroxide derived myoglobin compound II at pH 5.2 Received: 25 September 2000 / Accepted: 22 August 2001 / Published online: 11 October 2001 Ó SBIC 2001 Abstract The biological conversions of O 2 and perox- ides to water as well as certain incorporations of oxygen atoms into small organic molecules can be catalyzed by metal ions in dierent clusters or cofactors. The catalytic cycle of these reactions passes through similar metal- based complexes in which one oxygen- or peroxide- derived oxygen atom is coordinated to an oxidized form of the catalytic metal center. In haem-based peroxidases or oxygenases the ferryl Fe IV O) form is important in compound I and compound II, which are two and one oxidation equivalents higher than the ferric Fe III ) form, respectively. In this study we report the 1.35 A Ê structure of a compound II model protein, obtained by reacting hydrogen peroxide with ferric myoglobin at pH 5.2. The molecular geometry is virtually unchanged compared to the ferric form, indicating that these reactive interme- diates do not undergo large structural changes. It is further suggested that at low pH the dominating com- pound II resonance form is a hydroxyl radical ferric iron rather than an oxo-ferryl form, based on the short hy- drogen bonding to the distal histidine 2.70 A Ê ) and the FeáááO distance. The 1.92 A Ê FeáááO distance is in agree- ment with an EXAFS study of compound II in horse- radish peroxidase. Keywords Myoglobin á Compound II á Haem á Hydroxyl radical á Oxygen activation Introduction The main function of myoglobin is storage and trans- portation of oxygen in muscles. It can also carry out the same reactions as many peroxidases, with the cofactor haem-b serving as the peroxide reaction site [1]. How- ever, the peroxidase activity is rather weak owing to lack of a conserved distal arginine found in the active site of haem peroxidases. The ferric myoglobin reacts with peroxide by a heterolytic cleavage of the oxygen-oxygen bond, forming water and a transient ferryl compound I type complex [in which the haem-oxo complex is two oxidation equivalents higher than the ferric Fe III ) form]. Compound I is subsequently reduced by one electron to compound II and ®nally to the ferric form with a second electron, as depicted in the Scheme 1. For myoglobin this oxidation is believed to depend on the action of the distal His64 working as an acid/ base catalyst. For the ferric myoglobin form this his- tidine is hydrogen bonded to the iron-coordinated water molecule. Some authors suggest a homolytic cleavage of the peroxide bond, generating compound II directly and a free hydroxyl radical [2, 3] that in turn can form other radicals. The myoglobin compound II state can also be reached directly by hydrogen peroxide oxidation of ferrous Fe II ) or oxy-myoglobin forms [4]. As with the peroxidases, myoglobin reacting with peroxides can oxidize various small molecules in a classical peroxidase fashion [1, 4, 5]. Myoglobin's ferryl forms are probably biologically relevant since concen- trations of hydrogen peroxide above 4 lM are normally found in serum. The concentration can reach 200 lM by the activation of poly-morphonuclear leukocytes in vitro, and high peroxide levels may also occur during reperfusion after ischemia [6]. It has been shown in site-directed mutants, by iso- tope labelling and by electron paramagnetic resonance EPR) of myoglobin, that the reaction with peroxides can induce several semi-stable radicals [3, 7, 8, 9], J Biol Inorg Chem 2002) 7: 299±304 DOI 10.1007/s007750100296 H.-P. Hersleth and B. Dalhus contributed equally to this work. K.K. Andersson &) Department of Biochemistry, University of Oslo, P.O. Box 1041 Blindern, 0316 Oslo, Norway E-mail: k.k.andersson@biokjemi.uio.no Tel.: +47-2285-6625 Fax: +47-2285-4443 H.-P. Hersleth á B. Dalhus á C.H. GoÈrbitz Department of Chemistry, University of Oslo, P.O. Box 1033 Blindern, 0315 Oslo, Norway