ORIGINAL ARTICLE Robert E. Berry Æ Xiao D. Ding Tatjana Kh. Shokhireva Æ Andrzej Weichsel William R. Montfort Æ F. Ann Walker Axial ligand complexes of the Rhodnius nitrophorins: reduction potentials, binding constants, EPR spectra, and structures of the 4-iodopyrazole and imidazole complexes of NP4 Received: 17 April 2003 / Accepted: 16 October 2003 / Published online: 13 December 2003 Ó SBIC 2003 Abstract Previously, we utilized 4-iodopyrazole (4IPzH) as a heavy atom derivative for the initial solution of the crystal structure of the nitrophorin from Rhodnius pro- lixus, NP1, where it was found to bind to the heme with the iodo group disordered in two positions. We have now determined the structure of the 4IPzH complex of NP4 at pH 7.5 and find that the geometry and bond lengths at the iron center are extremely similar to those of the imidazole (ImH) complex of the same protein (structure determined at pH 5.6), except that the G–H loop is not in the closed conformation. 4IPzH binds to the heme of NP4 in an ordered manner, with the iodo substituent pointed toward the opening of the heme pocket, near the surface of the protein. In order to understand the solution chemistry in terms of the rela- tive binding abilities of 4IPzH, ImH, and histamine (Hm, a physiological ligand for the nitrophorins), we have also investigated the equilibrium binding constants and reduction potentials of these three ligand complexes of the four Rhodnius nitrophorins as a function of pH. We have found that, unlike the other Lewis bases, 4IPzH forms less stable complexes with the Fe(III) than the Fe(II) oxidation states of NP1 and NP4, and similar stability for the two oxidation states of NP2 and NP3, suggesting that this ligand is a softer base than ImH or Hm, for both of which the Fe(III) complexes are more stable than those of Fe(II) for all four nitrophorins. Surprisingly, in spite of this and the much lower basicity of 4IPzH than imidazole and histamine, the EPR g-values of all three ligand complexes are very similar. Keywords Histamine Æ Imidazole Æ 4-Iodopyrazole Æ Nitrophorin Æ Rhodnius Abbreviations NP1–4 nitrophorins 1–4 from Rhodnius prolixus Æ 4IPzH 4-iodopyrazole Æ ImH imidazole Æ Hm histamine Æ NO nitric oxide Æ NOS nitric oxide synthase Introduction The nitrophorins (nitro=NO, phorin=carrier) are a group of NO-carrying heme proteins found in the saliva of at least two species of blood-sucking insects, Rhodnius prolixus, the ‘‘kissing bug,’’ which has four such proteins [1], and Cimex lectularius, the bedbug, which has one ([2], Weichsel A, Maes EM, Andersen JF, Valenzuela JG, Berry RE, Shokhireva TK, Walker FA, Montfort WR, submitted). These interesting heme proteins sequester nitric oxide that is produced by a nitric oxide synthase (NOS) that is similar to vertebrate constitutive NOS, which is present in the cells of the salivary glands [3, 4, 5]. The NO is kept stable for long periods of time in the salivary glands by being bound as an axial ligand to the ferriheme centers of the nitrophorin proteins [6]. Upon injection into the tissues of the victim, NO dis- sociates, diffuses through the tissues to the nearby cap- illaries to cause vasodilation, and thereby allows more blood to be transported to the site of the wound. At the same time, in the case of the Rhodnius proteins, hista- mine, whose role is to cause swelling, itching, and the beginning of the immune response, is released by mast cells and platelets of the victim. This histamine binds to the heme sites of the Rhodnius nitrophorins, hence pre- venting the insectÕs detection for a period of time [7]. The Rhodnius proteins, which have been named NP1– 4 in order of their abundances in the insect saliva (NP1 most abundant), have been investigated by a number of spectroscopic techniques ([1, 8, 9, 10, 11, 12, 13], Shokhireva TKh, Smith KM, Andersen JF, Weichsel A, Balfour CA, Montfort WR, Walker FA, submitted) and the solid-state structures of one or more ligand J Biol Inorg Chem (2004) 9: 135–144 DOI 10.1007/s00775-003-0505-0 R. E. Berry Æ X. D. Ding Æ T. K. Shokhireva Æ F. A. Walker (&) Department of Chemistry, University of Arizona, Tucson, AZ 85721, USA E-mail: awalker@u.arizona.edu Tel.: +1-520-6218645 Fax: +1-520-6269300 A. Weichsel Æ W. R. Montfort Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ 85721, USA