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A study of the magnetic properties of haem a3 in cytochrome c oxidase by using magnetic-circular-dichroism spectroscopy.

ABSTRACT: M.c.d. (magnetic-circular-dichroism) spectroscopy was used to study the magnetization properties of the haem centres in cytochrome c oxidase with magnetic fields of between 0 and 5.3 T over the temperature range 1.5--200 K. The oxidized, oxidized cyanide and partially reduced cyanide forms of the enzyme were studied. In the oxidized state only cytochrome a3+ is detectable by m.c.d. spectroscopy, and its magnetization characteristics show it to be a low-spin ferric haem. In the partially reduced cyanide form of the enzyme cytochrome a is in the diamagnetic low-spin ferrous form, whereas cytochrome a3--CN is e.p.r.-detectable and gives an m.c.d.-magnetization curve typical of a low-spin ferric haem. In the oxidized cyanide form of the enzyme both cytochrome a and cytochrome a3--CN are detectable by m.c.d. spectroscopy, although only cytochrome a gives an e.p.r. signal. The magnetization characteristics of haem a3--CN show clearly that its ground state is an electronic doublet and that another state, probably a spin singlet, lies greater than 10 cm-1 above this. These features are well accounted for by an electronic state of spin S = 1 with a predominantly axial distortion, which leaves the doublet, Ms = +/- 1, as the ground state and the component Ms = 0 as the excited state. This state would not give an e.p.r. signal. Such an electronic state could arise either from a ferromagnetic coupling between haem a3+(3)-CN and the cupric ion, Cua3, or form a haem in the Fe(IV) state.

SUBMITTER: Thomson AJ

PROVIDER: S-EPMC1162656 | biostudies-other | 1981 Mar

REPOSITORIES: biostudies-other

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Project description:Although extensive research has been performed on various cytochrome P450s, especially Cyt P450cam, there is much to be learned about the mechanism of how its functional unit, a heme b ligated by an axial cysteine, is finely tuned for catalysis by its second coordination sphere. Here we study how the hydrogen-bonding network affects the proximal cysteine and the Fe-S(Cys) bond in ferric Cyt P450cam. This is accomplished using low-temperature magnetic circular dichroism (MCD) spectroscopy on wild-type (wt) Cyt P450cam and on the mutants Q360P (pure ferric high-spin at low temperature) and L358P where the "Cys pocket" has been altered (by removing amino acids involved in the hydrogen-bonding network), and Y96W (pure ferric low-spin). The MCD spectrum of Q360P reveals fourteen electronic transitions between 15200 and 31050 cm(-1). Variable-temperature variable-field (VTVH) saturation curves were used to determine the polarizations of these electronic transitions with respect to in-plane (xy) and out-of-plane (z) polarization relative to the heme. The polarizations, oscillator strengths, and TD-DFT calculations were then used to assign the observed electronic transitions. In the lower energy region, prominent bands at 15909 and 16919 cm(-1) correspond to porphyrin (P) → Fe charge transfer (CT) transitions. The band at 17881 cm(-1) has distinct sulfur S(π) → Fe CT contributions. The Q band is observed as a pseudo A-term (derivative shape) at 18604 and 19539 cm(-1). In the case of the Soret band, the negative component of the expected pseudo A-term is split into two features due to mixing with another π → π* and potentially a P → Fe CT excited state. The resulting three features are observed at 23731, 24859, and 25618 cm(-1). Most importantly, the broad, prominent band at 28570 cm(-1) is assigned to the S(σ) → Fe CT transition, whose intensity is generated through a multitude of CT transitions with strong iron character. For wt, Q360P, and L358P, this band occurs at 28724, 28570, and 28620 cm(-1), respectively. The small shift of this feature upon altering the hydrogen bonds to the proximal cysteine indicates that the role of the Cys pocket is not primarily for electronic fine-tuning of the sulfur donor strength but is more for stabilizing the proximal thiolate against external reactants (NO, O(2), H(3)O(+)), and for properly positioning cysteine to coordinate to the iron center. This aspect is discussed in detail.

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A study of the magnetic properties of haem a3 in cytochrome c oxidase by using magnetic-circular-dichroism spectroscopy.

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