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Kinetic and spectroscopic studies of haemoglobin and myoglobin from Urechis caupo. Distal residue effects.

ABSTRACT: Seven components of the tetrameric haemoglobin (Hbu) from Urechis caupo were separated by preparative isoelectric focusing and characterized by their absorption spectra and pI values. The helix content and Soret delta epsilon values are reported for several of the components. Temperature-jump O2-binding kinetics of the major components of Hbu show biphasic behaviour, with the majority species having kon = 1.57 x 10(9) mol-1.s-1 and koff = 3.32 x 10(4) s-1. The Fourier-transform i.r. spectrum of pooled Hbu(II)-CO displays a stretching frequency of 1942 cm-1. E.s.r. of Hbu(II)-NO demonstrates evidence of proximal strain similar to that encountered in T-state human haemoglobin. CO-driven reduction of U. caupo methaemoglobin, Hbu(III) and U. caupo metmyoglobin [Mbu(III)] shows much higher rates relative to haemoglobins and myoglobins known to possess a distal histidine residue. Nitrosyl auto-reduction kinetics of Hbu(III)-NO and Mbu(III)-NO are examined. The equilibrium binding constants of several ligands are reported for both Hbu and Mbu, and together with the above kinetic data suggest differences in haem pocket environments between Hbu and Mbu. Reaction of Hbu with 2-chloromercuri-4,6-dinitrophenol demonstrates the presence of one reactive thiol group per globin chain. lambda max. values and the respective molar absorption coefficients for selected ligand-bound states are reported for the major component of Hbu and for Mbu. The majority haem orientation in U. caupo haemoglobin is identical with that of human haemoglobin.

SUBMITTER: DiFeo TJ

PROVIDER: S-EPMC1131650 | biostudies-other | 1990 Aug

REPOSITORIES: biostudies-other

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Project description:It is now well-established that mammalian heme proteins are reactive with various nitrogen oxide species and that these reactions may play significant roles in mammalian physiology. For example, the ferrous heme protein myoglobin (Mb) has been shown to reduce nitrite (NO(2)(-)) to nitric oxide (NO) under hypoxic conditions. We demonstrate here that the distal pocket histidine residue (His64) of horse heart metMb(III) (i.e., ferric Mb(III)) has marked effects on the mode of nitrite ion coordination to the iron center. X-ray crystal structures were determined for the mutant proteins metMb(III) H64V (2.0 A resolution) and its nitrite ion adduct metMb(III) H64V-nitrite (1.95 A resolution), and metMb(III) H64V/V67R (1.9 A resolution) and its nitrite ion adduct metMb(III) H64V/V67R-nitrite (2.0 A resolution). These are compared to the known structures of wild-type (wt) hh metMb(III) and its nitrite ion adduct hh metMb(III)-nitrite, which binds NO(2)(-) via an O-atom in a trans-FeONO configuration. Unlike wt metMb(III), no axial H(2)O is evident in either of the metMb(III) mutant structures. In the ferric H64V-nitrite structure, replacement of the distal His residue with Val alters the binding mode of nitrite from the nitrito (O-binding) form in the wild-type protein to a weakly bound nitro (N-binding) form. Reintroducing a H-bonding residue in the H64V/V67R double mutant restores the O-binding mode of nitrite. We have also examined the effects of these mutations on reactivities of the metMb(III)s with cysteine as a reducing agent and of the (ferrous) Mb(II)s with nitrite ion under anaerobic conditions. The Mb(II)s were generated by reduction of the Mb(III) precursors in a second-order reaction with cysteine, the rate constants for this step following the order H64V/V67R > H64V >> wt. The rate constants for the oxidation of the Mb(II)s by nitrite (giving NO as the other product) follow the order wt > H64V/V67R >> H64V and suggest a significant role of the distal pocket H-bonding residue in nitrite reduction.

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Kinetic and spectroscopic studies of haemoglobin and myoglobin from Urechis caupo. Distal residue effects.

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