Cross-polarization/magic-angle sample-spinning ¹³ C NMR spectroscopic study of chlorophyll a in the solid state

Abstract

Solid-state cross-polarization/magic-angle sample-spinning 13C NMR spectra have been recorded on chlorophyll a-water aggregates, methyl pyrochlorophyllide a, and methyl pyropheophorbide a (derivatives that lack a phytyl chain). Spectra have also been collected under a decoupling regime in which resonances of certain hydrogen-bearing carbon atoms are suppressed. These observations are used to assign the solid-state spectra.High-resolution NMR spectroscopy has made very important contributions to the understanding of chlorophyll function at the molecular level. Not only has NMR been useful in establishing the identity of several chlorophylls of previously unknown structure but it has also provided new insights on the coordination donor-acceptor properties of the chlorophylls. The latter has contributed significantly to a better understanding of the state of chlorophyll in vivo. The contributions of NMR to chlorophyll studies have been the subject of a recent comprehensive review (1). To acquire high-resolution NMR spectra it has been necessary up to now that these be recorded in the liquid state or in solution, where the chlorophylls under investigation have high mobility and can rotate freely. However, there are good reasons to suppose that chlorophyll in vivo is largely immobilized, and direct application of high-resolution NMR by conventional techniques to the state of chlorophyll in vivo has not been practical. New developments in NMR theory and instrumentation now make it possible to obtain useful, high-resolution NMR spectra on immobilized (solid) samples (2, 3). Therefore, we have undertaken an investigation of the NMR spectroscopy of chlorophyll in the solid state, with a view to acquiring the basic information necessary for the future applications of solid-state NMR spectroscopy to in vivo or in vivo-like chlorophyll systems.METHODS Cross-polarization/magic-angle sample-spinning (CP/MAS) 13C NMR spectra of solid samples were recorded on Nicolet S-100 and NT-150 spectrometers. The samples (50-500 mg) were prepared as fine powders, which were loosely packed in the rotors. Rotors were spun at the magic angle (54044' from the direction of the magnetic field, BO) at rates of 2.5-2.9 kHz. Because of technical problems in confining samples of material that flow when spun at high speeds, we have restricted our investigation to hydrated chlorophyll a (Chl a) and to chlorophyll derivatives in which the phytyl chains have been removed (Fig. 1). Dipolar broadening by protons was further reduced by simultaneous irradiation with 11 G (1 G = 10-4 T) of decoupling field. Spectra like those in Fig. 2 were recorded with only 50 mg of sample by pulsing overnight.To facilitate spectral assignments, signals from carbon atoms to which hydrogen atoms are attached were selectively suppressed. This is accomplished by inserting a 40-gsec delay without radiofrequency fields between the mix and acquisition periods of the cross polarization pulse sequence (4). Carbons that experience strong dipolar coupling to bonded protons ...

Magneto-Optic Spectroscopy of a Protein Tetramer Binding Two Exciton-Coupled Chlorophylls

“…This interaction is typically bridged by a nucleophilic molecule(s), such as water, that is able to both ligate to the Mg and hydrogen bond to the carbonyl groups. The details of the aggregation interaction and relative Chl orientations appear to be different in the solid state of Chl−water aggregates ,,, than for Chl dimers in solution. ,− ,,,,, In the absence of extraneous nucleophiles able to coordinate to the Mg, Chl a aggregation may occur via direct interaction of the 13 1 carbonyl group with the Mg of a neighboring Chl a . ,− ,,, …”