Chien-wei Li

Publication Date


Document Type


First Advisor

Baker, Gary M.

Degree Name

M.S. (Master of Science)

Legacy Department

Department of Chemistry


Cytochrome oxidase; Hydrogen-ion concentration; Absorption spectra


The effect of pH on the near-uv absorption spectrum of cytochrome oxidase has been examined. Several lines of evidence are consistent with a proton binding site that can modulate the optical properties of cytochrome a^. Changing the pH within the range 6.7 to 10.5 was found to reversibly shift the position of the Soret band over an 11 nm range. The lower pH values caused a progressive blue shift in the Soret band, whereas the high pH range promoted a gradual red shift. Limiting band positions were 416 and 427 nm, respectively. The incubation time required to produce these extreme band positions varied, but in all cases an isosbestic point was evident. Magnetic circular dichroism (MCD) of the low pH form at 416 nm, the high pH form at 427 nm, and the cyanide derivative at 428 nm were compared. No evidence of a high pH-dependent low spin transition or a change in the redox state of cytochrome a3 was found, confirming earlier work by Baker, G.M., Naguchi, M., and Palmer, G. (1987) J. Biol. Chem. 262, 595-604. Subtraction of cytochrome a (spectrum taken from Vanneste, W.H. (1966) Biochemistry 5, 838-848) from a series of blue shifting spectra showed a band at 414 nm that progressively gained amplitude and a band at 430 nm that correspondingly lost amplitude. A series of red shifting spectra showed the opposite behavior with a clear isosbestic point being evident in both cases. The 430 nm band is noted to be unusually red-shifted for high spin, ferric heme a. The pH titration profiles for the apparent equilibrium position of the Soret band at different heme a concentrations were obtained. Nonlinear least squares fitting, assuming a single acid/base group, showed that the pKa was dependent on heme a concentration. Various explanations for the pH-dependent shift in the near-uv absorption profiles of cytochrome a3 are discussed.


Includes bibliographical references (pages [58]-61)


61 pages




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