Stephen P. A. Fodor

Resonance Raman spectra of the Pr-form of phytochrome

Abstract— Resonance Raman spectra of the Pr‐form of oat phytochrome have been obtained at 77 K. Interference from phytochrome fluorescence is avoided by employing far‐red 752 nm excitation. Vibrational assignments are suggested for the tetrapyrrole chromophore in phytochrome by comparison with previously published model compound spectra and by examining the characteristic shifts induced by deuteration of the pyrrole nitrogens. The lack of carbonyl intensity, the frequencies of the 1626 and 1644 cm‐1 C=C stretching modes, and the presence of an intense mode at 1326 cm‐1 are all consistent with a protonated structure for the tetrapyrrole chromophore in Pr. This suggests that the ‐50 nm red‐shift of the protein‐bound chromophore absorption compared to the chromophore in vitro is caused by protonation of the pyrrole nitrogen.

RESONANCE RAMAN SPECTRA OF BACTERIORHODOPSIN MUTANTS WITH SUBSTITUTIONS AT ASP‐85, ASP‐96, AND ARG‐82

Detergent solubilized bacteriorhodopsin (BR) proteins which contain alterations made by site‐directed mutagenesis (Asp‐96→Asn, D96N; Asp‐85→Asn, D85N; and Arg‐82→Gln, R82Q) have been studied with resonance Raman spectroscopy. Raman spectra of the light‐adapted (BRLA) and M species in D96N are identical to those of native BR, indicating that this residue is not located near the chromophore. The BRLA states of D85N and especially R82Q contain more of the 13‐cis, C=N syn (BR555) species under ambient illumination compared to solubilized native BR. Replacement of Asp‐85 with Asn causes a 25 nm red‐shift of the absorption maximum and a frequency decrease in both the ethylenic (‐7 cm−1) and the Schiff base C=NH+ (‐3 cm−1) stretching modes of BRLA. These changes indicate that Asp‐85 is located close to the protonated retinal Schiff base. The BRLA spectrum of R82Q exhibits a slight perturbation of the C=NH+ band, but its M spectrum is unperturbed. The Raman spectra and the absorption properties of D85N and R82Q suggest that the protein counterion environment involves the residues Asp‐85−, Arg‐82+ and presumably Asp‐212−. These data are consistent with a model where the strength of the protein‐chromophore interaction and hence the absorption maximum depends on the overall charge of the Schiff base counterion environment.

ULTRAVIOLET RESONANCE RAMAN SPECTROSCOPY OF BACTERIORHODOPSIN

Abstract— Ultraviolet resonance Raman spectra of bacteriorhodopsin have been obtained using 229 nm excitation from a hydrogen‐shifted neodymium yttrium aluminum garnet (Nd: YAG) laser. High signal‐to‐noise spectra are observed exhibiting vibrational bands at 762, 877, 1011, 1175, 1356, 1552 and 1617 cm−1 which are assigned to scattering from tryptophan and tyrosine side chains. This demonstrates the feasibility of using UV resonance Raman spectroscopy to monitor aromatic amino acid structural changes during the bacteriorhodopsin photocycle.