title: Time-resolved absorption and magnetic circular dichroism spectroscopy of cytochrome c3 from Desulfovibrio.
abstract: The UV-visible absorption and magnetic circular dichroism (MCD) spectra of the ferric, ferrous, CO-ligated forms and kinetic photolysis intermediates of the tetraheme electron-transfer protein cytochrome c3 (Cc3) are reported. Consistent with bis-histidinyl axial coordination of the hemes in this Class III c-type cytochrome, the Soret and visible region MCD spectra of ferric and ferrous Cc3 are very similar to those of other bis-histidine axially coordinated hemeproteins such as cytochrome b5. The MCD spectra indicate low spin state for both the ferric (S = 1/2) and ferrous (S = 0) oxidation states. CO replaces histidine as the axial sixth ligand at each heme site, forming a low-spin complex with an MCD spectrum similar to that of myoglobin-CO. Photodissociation of Cc3-CO (observed photolysis yield = 30%) produces a transient five-coordinate, high-spin (S = 2) species with an MCD spectrum similar to deoxymyoglobin. The recombination kinetics of CO with heme Fe are complex and appear to involve at least five first-order or pseudo first-order rate processes, corresponding to time constants of 5.7 microseconds, 62 microseconds, 425 microseconds, 2.9 ms, and a time constant greater than 1 s. The observed rate constants were insensitive to variation of the actinic photon flux, suggesting noncooperative heme-CO rebinding. The growing in of an MCD signal characteristic of bis-histidine axial ligation within tens of microseconds after photodissociation shows that, although heme-CO binding is thermodynamically favored at 1 atm CO, binding of histidine to the sixth axial site competes kinetically with CO rebinding.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064.
authors.forename: D B
authors.initials: DB
authors.lastname: O'Connor
authors.forename: R A
authors.initials: RA
authors.lastname: Goldbeck
authors.forename: J H
authors.initials: JH
authors.lastname: Hazzard
authors.forename: D S
authors.initials: DS
authors.lastname: Kliger
authors.forename: M A
authors.initials: MA
authors.lastname: Cusanovich
mesh_terms: D055592:Biophysical Phenomena; D001703:Biophysics; D002248:Carbon Monoxide / Q000737:chemistry; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000528:radiation effects; D016969:Desulfovibrio vulgaris / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D008280:Magnetics; D010782:Photolysis; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
chemical_list: D003574:Cytochrome c Group; D006418:Heme; D002248:Carbon Monoxide; C017165:cytochrome c(3)