The use of principal component analysis to resolve the spectra and kinetics of cytochrome c oxidase reduction by 5,10-dihydro-5-methyl phenazine

Abstract

The method of principal component analysis (PCA) was applied to the absorption-wavelength-time surfaces generated by rapid scanning stopped-flow spectrophotometry (RSSFS). The method was used to resolve the absorption surfaces generated during the reduction of cytochrome c oxidase by 5,10-dihydro-5-methyl phenazine (MPH) into the individual spectral shapes and time courses of the component chromophores. Two forms of resting cytochrome oxidase were used in these analyses: one that has its maximum absorption in the Soret region at 418 nm (418-nm species) and the other has its absorption maximum at 424 nm (424-nm species). A weighting scheme suitable for RSSFS data was developed. The optical absorption spectra obtained by W.H. Vanneste (1966, Biochemistry, 5:838-848) for the oxidase components were found to fit adequately as components of the experimental surfaces. Among these spectra were the oxidized forms of cytochromes a and a3 in the wavelength region 330-520 nm for the 418-nm species. Vanneste's spectral shape for the oxidized cytochrome a3 did not fit as a component in the spectrum of the 424-nm species. After accounting for the spectral shape of all components present, PCA provided a straightforward method for determining the separate time courses of each chromophore. We have found for both forms used that cytochrome a is reduced by MPH in the initial stages of the reaction, while cytochrome a3 is reduced in subsequent, slow phases. An important aspect of PCA is that it provided confirmation of the spectra of the various oxidase components without requiring the use of inhibitors or the use of simplifying mechanistic assumptions. The resolution of time profiles of strongly overlapping chromophores is also demonstrated.

Formation of Stable BOBO-3 H-Aggregate Complexes Hinders DNA Hybridization

“…It is therefore more correct to use the term "absorber" to allude to species in PCA. 47 The fact that both the H-aggregate complex and the electrostatically driven interactions of BOBO-3 behave as a single absorber confirms that their concentration grows proportionally to each other as a function of (Olig6C) 30 concentration. This suggests that both types of interactions have comparable affinity constants to each other.…”

Effect of carbon dioxide on self-setting apatite cement formation from tetracalcium phosphate and dicalcium phosphate dihydrate; ATR-IR and chemoinformatics analysis

“…Principal component analysis (PCA) is a multivariate analysis that can be used for the analyses of reaction kinetics and of spectra obtained by spectroscopies [14].…”

Thermodynamic studies of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast microsomes

“…This method, also known as bilinear factor analysis [26] or singular value decomposition (SVD) analysis [27], was first introduced in chemistry by Malinowski and Howery [28]. It is widely used nowadays to interpret absorbance and fluorescence spectral changes in complex biochemical systems [26][27][28][29][30][31][32]. Using this approach we have deduced the set of standard absorbance spectra of the high-spin, low-spin and P-420 states of pure ferric microsomal cytochrome P-450 2B4 from rabbit liver.…”