The Second Derivative Electronic Absorption Spectrum of Cytochrome c Oxidase in the Soret Region

Abstract

The electronic absorption spectrum of solubilized beef heart cytochrome c oxidase was analyzed in the 400-500 nm region to identify the origin of doublet features appearing in the second derivative spectrum associated with ferrocytochrome a. This doublet, centered near 22,600 cm(-1), was observed in the direct absorption spectrum of the a(2+)a(3)(3+).HCOO(-) form of the enzyme at cryogenic temperatures. Since evidence for this doublet at room temperature is obtained only on the basis of the second derivative spectrum, a novel mathematical approach was developed to analyze the resolving power of second derivative spectroscopy as a function of parameterization of spectral data. Within the mathematical limits defined for resolving spectral features, it was demonstrated that the integrated intensity of the doublet feature near 450 nm associated with ferrocytochrome a is independent of the ligand and oxidation state of cytochrome a(3). Furthermore, the doublet features, also observed in cytochrome c oxidase from Paracoccus denitrificans, were similarly associated with the heme A component and were correspondingly independent of the ligand and oxidation state of the heme A(3) chromophore. The doublet features are attributed to lifting of the degeneracy of the x and y polarized components of the B state of the heme A chromophore associated with the Soret transition.

Insight into co-operative growth of nearly monodispersive CdS nanocrystals embedded in polyvinyl pyrrolidone

“…2. Band gap (Eg) is obtained by calculating the second derivative [35,36] of the absorption spectrum having peak *2.68 eV (462 nm). The band gap from PL spectrum is obtained to be *2.65 eV (468 nm, FWHM *30 nm) by fitting the PL peak (lowest) to a Gaussian function.…”

A general method of computing the derivative of experimental data

“…The discrete points in Figure 3a are the absorption spectrum A M (ν) of cytochrome c oxidase digitized from a spectrum reported by Horvath et al,10 N D = 332 in this digitized version of the spectrum. The second‐ and first‐derivative spectra given by Eq.…”

A General Computational Method for Converting Normal Spectra into Derivative Spectra

“…Horvath et al analyzed the visible absorption and second-derivative spectra of cytochrome c oxidase under different thermal and chemical conditions. 11 They have also investigated the numerical performance of the SG method. In their investigation they plotted the absorbance of carbon-monoxide bound cytochrome c oxidase as a function of wave number in the range 20 500 cm Ϫ1 Յ Յ 25 500 cm Ϫ1 and ⌬ ϭ 10 cm Ϫ1 .…”

“…Thus, there are around 500 points in the as-measured spectrum. Figure 4a is an enlarged, scanned, and digitized version of the continuous A M () curve taken from Horvath et al 11 In this particular digitizing there are 332 points in the resulting data file. Thus the N D used in Eq.…”