Intermediate spectra and photocycle kinetics of the Asp96 → Asn mutant bacteriorhodopsin determined by singular value decomposition with self-modeling

Singular value decomposition with self-modeling is applied to resolve the intermediate spectra and kinetics of the Asp96 → Asn mutant bacteriorhodopsin. The search for the difference spectra of the intermediates is performed in eigenvector space on the stoichiometric plane. The analysis of data at pH values ranging from 4 to 8 and temperatures between 5 and 25°C reveals significant, early partial recovery of the initial state after photoexcitation. The derived spectra are not biased by assumed photocycles. The intermediate spectra derived in the initial step differ from spectra determined in prior analyses, which results in intermediate concentrations with improved stoichiometric properties. Increasingly more accurate photocycles follow with increasing assumed complexity, of which parallel models are favored, consistent with recent, independent experimental evidence.