Preparation and spectral characterization of the heme d1.apomyoglobin complex: an unusual protein environment for the substrate-binding heme of Pseudomonas cytochrome oxidase.
The heme d1 prosthetic group isolated from Pseudomonas cytochrome oxidase combines with apomyoglobin to form a stable, optically well-defined complex. Addition of ferric heme d1 quenches apomyoglobin tryptophan fluorescence suggesting association in a 1:1 molar ratio. Optical absorption maxima for heme d1.apomyoglobin are at 629 and 429 nm before, and 632 and 458 nm after dithionite reduction; they are distinct from those of heme d1 in aqueous solution but more similar to those unobscured by heme c in Pseudomonas cytochrome oxidase. Cyanide, carbon monoxide and imidazole alter the spectrum of heme d1.apomyoglobin demonstrating axial coordination to heme d1 by exogeneous ligands. The cyanide-induced optical difference spectra exhibit isosbestic points, and a Scatchard-like analysis yields a linear plot with an apparent dissociation constant of 4.2 X 10(-5) M. However, carbon monoxide induces two absorption spectra with Soret maxima at 454 or 467 nm, and this duplicity, along with a shoulder that correlates with the latter before binding, suggests multiple carbon monoxide and possibly heme d1 orientations within the globin. The 50-fold reduction in cyanide affinity over myoglobin is more consistent with altered heme pocket interactions than the intrinsic electronic differences between the two hemes. However, stability of the heme d1.apomyoglobin complex is verified further by the inability to separate heme d1 from globin during dialysis and column chromatography in excess cyanide or imidazole. This stability, together with a comparison between spectra of ligand-free and -bound derivatives of heme d1-apomyoglobin and heme d1 in solution, implies that the prosthetic group is coordinated in the heme pocket through a protein-donated, strong-field ligand. Furthermore, the visible spectrum of heme d1.apomyoglobin varies minimally with ligand exchange, in contrast to the Soret, which suggests that much spectral information concerning heme d1 coordination in the oxidase is lost by interference from heme c absorption bands. A comparison of the absorption spectra of heme d1.apomyoglobin and Pseudomonas cytochrome oxidase, together with a critical examination of the previous axial ligand assignments from magnetic resonance techniques in the latter, implies that it is premature to accept the assignment of bishistidine heme d1 coordination in oxidized, ligand-free oxidase and other iron-isobacteriochlorin-containing enzymes.
Steup
M B
MB
Department of Chemistry, Purdue University School of Science, Indiana University-Purdue University, Indianapolis.
Muhoberac
B B
BB
D001059:Apoproteins / Q000378:metabolism*; D002248:Carbon Monoxide; D003486:Cyanides / Q000378:metabolism; D004227:Dithionite; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D008024:Ligands; D009211:Myoglobin / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology*; D012996:Solutions; D013053:Spectrophotometry
D001059:Apoproteins; D003486:Cyanides; D008024:Ligands; D009211:Myoglobin; D012996:Solutions; C010857:apomyoglobin; D004227:Dithionite; D006418:Heme; D002248:Carbon Monoxide; D003576:Electron Transport Complex IV