Calculated vibrational properties of pigments in protein binding sites 2: Semiquinones in photosynthetic proteins.

[QA- - QA] Fourier transform infrared difference spectra have previously been obtained using purple bacterial reaction centers from Rhodobacter sphaeroides with unlabeled, 18O and 13C isotope labeled phylloquinone (PhQ, also known as vitamin K1) incorporated into the QA protein binding site (Breton, (1997), Proc. Natl. Acad. Sci. USA94 11318-11323). The nature of the bands in these spectra and the isotope induced band shifts are poorly understood, especially for the phyllosemiquinone anion (PhQ-) state. To aid in the interpretation of the bands in these experimental spectra, ONIOM type QM/MM vibrational frequency calculations were undertaken. Calculations were also undertaken for PhQ- in solution. Surprisingly, both sets of calculated spectra are similar and agree well with the experimental spectra. This similarity suggests pigment-protein interactions do not perturb the electronic structure of the semiquinone in the QA binding site. This is not found to be the case for the neutral PhQ species in the same protein binding site. PhQ also occupies the A1 protein binding site in photosystem I, and the vibrational properties of PhQ- in the QA and A1 binding sites are compared and shown to exhibit considerable differences. These differences probably arise because of changes in the degree of asymmetry of hydrogen bonding of PhQ- in the A1 and QA binding sites.

Comparison of calculated and experimental isotope edited FTIR difference spectra for purple bacterial photosynthetic reaction centers with different quinones incorporated into the QA binding site.

Previously we have shown that ONIOM type (QM/MM) calculations can be used to simulate isotope edited FTIR difference spectra for neutral ubiquinone in the QA binding site in Rhodobacter sphaeroides photosynthetic reaction centers. Here we considerably extend upon this previous work by calculating isotope edited FTIR difference spectra for reaction centers with a variety of unlabeled and (18)O labeled foreign quinones incorporated into the QA binding site. Isotope edited spectra were calculated for reaction centers with 2,3-dimethoxy-5,6-dimethyl-1,4-benzoquinone (MQ0), 2,3,5,6-tetramethyl-1, 4-benzoquinone (duroquinone, DQ), and 2,3-dimethyl-l,4-naphthoquinone (DMNQ) incorporated, and compared to corresponding experimental spectra. The calculated and experimental spectra agree well, further demonstrating the utility and applicability of our ONIOM approach for calculating the vibrational properties of pigments in protein binding sites. The normal modes that contribute to the bands in the calculated spectra, their composition, frequency, and intensity, and how these quantities are modified upon (18)O labeling, are presented. This computed information leads to a new and more detailed understanding/interpretation of the experimental FTIR difference spectra. Hydrogen bonding to the carbonyl groups of the incorporated quinones is shown to be relatively weak. It is also shown that there is some asymmetry in hydrogen bonding, accounting for 10-13 cm(-1) separation in the frequencies of the carbonyl vibrational modes of the incorporated quinones. The extent of asymmetry in H-bonding could only be established by considering the spectra for various types of quinones incorporated into the QA binding site. The quinones listed above are "tail-less." Spectra were also calculated for reaction centers with corresponding "tail" containing quinones incorporated, and it is found that replacement of the quinone methyl group by a phytyl or prenyl chain does not alter ONIOM calculated spectra.

Viral infection of cells in culture detected using infrared microscopy.

FTIR microscopy has been used to collect spectra for uninfected (mock) Vero cells, and cells that have been infected with herpes simplex virus type 1 (HSV-1) and human adenovirus type 5 (Ad-5). Cells were infected at a multiplicity of infection of 10, and studied at 24 hours post exposure. The spectra for infected samples display many differences compared to the spectra for uninfected samples. To estimate how well the spectra for uninfected and infected samples could be discriminated, we used logistic and partial least squares regression methods. We show that the spectra for HSV-1 and mock infected samples are well differentiated and, for a sensitivity of 95%, we calculate a specificity of 0.999 using partial least squares regression. Spectra for Ad-5 and mock infected samples are also well differentiated. We find that applying our regression models constructed with one data set to a new validating data set still gives very high levels of specificity for a given sensitivity. Spectra for Ad-5 and HSV-1 infected samples are also differentiable. Applying our constructed regression models to new validating data, however, leads to a decrease in the discrimination capability in this instance. If one is simply interested in differentiating spectra associated with uninfected and infected cells, without distinguishing the type of infection, then we show that logistic regression models can break down whereas partial least squares regression models perform well.