Volume 314, number 3,481485 FEES 1192i Q 1992 Federation of European Biochemical Societies 00145793/92/S5.00 December 1992 The homodimeric hemoglobin from zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF Scapharca can be locked into new cooperative structures upon reaction of Cys”, located at the subunit interface, with organomercurials Gianni Colotti”? Albert0 Boffi”, Daniela Verzili”, Massimiliano Colettab and Emilia Chianconc” “CNR Center of Molertdar Riology, Departmnr of Biochemical 3ciences ‘A. Rossi FuneW , University ‘La Sapienza’, 00185 Rome, Italy and “Department of Mofecldar. Celltdar und Artimal Biology, University of Cutnerino. Via I;: Camerini, 2, 62032 Camrrino (M C), Italy Received 9 November 1992 In the coopcrallve, homodimcric hemoglobin from Scu~~hcrr~crr irrrteq~risatris, HbI, the subunit interface is formL4 by the hemc-carrying E and F helices and contnins he only cysreine residue of the globin chnin (Cys”‘, F2) in an area which changes from hydrophilic to hydrophobic upon oxygenation. Binding of organomercurials to Hbl is cooperntivc und entails major qualernary rcarrangemcnls. The reaction of Cys” with p-chloron7ercuri-benzoate (PMB) and p-niuo-o-chloromercuriphenol (PN), a senshivc reporter of the cysteine microenvironment zyxwvutsrqponmlkjihgf al neulr;ll pH values, has been followed in stopped flow experiments. Kinclic evidence for the cooperntivity of mercurial binding has been obtained and the rate of the corresponding confnrmiltional transition has been estimated. As expected PN. but 1101 PMB, is able to monitor the oxygen-linked change of the cysteinc microenvironment. The modification of Cy@ with PN has unique functional effects. In PN-reacled Hbl cooperativity is maintained, albch to a different extent, depending on the ligation state of the protein during mcrcnptidc formation. II may be envisaged that PN locks the protein into new, cooperative, quaternary structures stabilized by hydrogen bonding interactions between the ionized nitrophcnol moiety and the contra- lateml subunit. Molluscun hemoglobin; Cooperntivity: Chemical modification; Conformational change 1. INTRODUCTION In the homodimeric hemoglobin, HbI, from the Arcid clam Scapharca zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA imeqthdvis cooperativity in oxygen binding [1] arises from a molecular mechanism which differs from that operative in tetrameric vertebrate he- moblogins. This peculiarity reflects the unique mode of assembly of the globin chains: in Hbl the heme-carrying E and F heliced do not Pace outwards, but form the subunit interface and bring the two heme groups in direct communication via a hydrogen bond network which differs in detail in the carbonmonoxy and deoxy derivatives [2,3]. At variance with vertebrate hemo- globins, oxygen binding is accompanied by only subtle quaternary movements of the two subunits relative to one another [33. The two symmetry-related regions of the subunit in- terface where the most significant ligand-linked changes occur contain CyPZ (F2). the only cysteine residue of the polypeptide chain [4]. The reactivily of Cys’” therefore provides an ideal handle to both study and affect the interface. Indeed Boffi et al. [5] l>bserved that sulfhydryl alter the oxygen binding properties of Mb1 and that the Correspothwe uctctfess: E. Ciliancone, CNR Ccniur of XuiWui&r Biology, c/o Dept. of Biochemical Sciences ‘A. Rossi Fanelli’, Univer- sity ‘La Sapicnza’, 00185 Rome, Italy. Fax: (39) (6) 444OOG2. effect depends to a first approximation on the size of the reagent zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM used. p-Chloromercuribenzoate (PMB), for ex- ample, brings about a significant increase in oxygen affinity accompanied by the loss of cooperativity. These functional properties reflect the drastic change in qua- ternary structure which causes Ihe HbI crystals to crack upon reaction with PMB. Boffi et al. [S] also disclosed an unexpected property of the two Cyd’ residues in oxy-Hbl, namely that they bind PMB in a cooperative fashion. The present paper reports a study of the reactivity of Cys9’ with a chromophoric mercurial, p-nitro-o-chlo- romercuriphenol (PN), and of the oxygen binding prop erties of the modified protein. PM is a sensitive reporter group of the cysteine microenvironment at neutral pH values due to the ionization of its nitrophenol group [6]. In principle therefore PN should allow one to monitor the oxygen-linked changes in hydrophilicity around Cys” attendant the movement of Phe97. Thus, the X-ray structures [2,3] show that in the liganded derivative the phenyl group of Pheg7 lies in the same hydrophobic patch of the interface that comprises Cys”, but leaves the interface and packs against the proximal His’“’ upon deoxygenation. The kinetics of Cys’? modification by PN depends on t!le state of oxygenation of the protein, provides evi- dence for cooperativity in mercurial binding and for the 481