Low-frequency Fourier transform infrared spectroscopy of the oxygen-evolving and quinone acceptor complexes in photosystem II

The low-frequency (<1000 cm(-1)) region of the IR spectrum has the potentia l to provide detailed structural and mechanistic insight into the photosyst em II/oxygen evolving complex (PSII/OEC). A cluster of four manganese ions forms the core of the OEC and diagnostic manganese-ligand and manganese-sub strate modes are expected to occur in the 200-900 cm(-1) range. However, wa ter also absorbs IR strongly in this region, which has limited previous Fou rier transform infrared (FTIR) spectroscopic studies of the OEC to higher f requencies (>1000 cm(-1)). We have overcome the technical obstacles that ha ve blocked FTIR access to low-frequency substrate, cofactor, and protein vi brational modes by using partially dehydrated samples, appropriate window m aterials, a wide-range MCT detector, a novel band-pass filter, and a closel y regulated temperature control system. With this design, we studied PSII/O EC samples that were prepared by brief illumination of O-2 evolving and Tri s-washed preparations at 200 K or by a single saturating laser flash applie d to O-2 evolving and inhibited samples at 250 K. These protocols allowed u s to isolate low-frequency modes that are specific to the Q(A)(-)/Q(A) and S-2/S-1 states. The high-frequency FTIR spectra recorded for these samples and parallel EPR experiments confirmed the states accessed by the trapping procedures we used. In the S-2/S-1 spectrum, we detect positive bands at 63 1 and 602 cm(-1) and negative bands at 850, 679, 664, and 650 cm(-1) that a re specifically associated with these two S states. The possible origins of these IR bands are discussed. For the low-frequency Q(A)(-)/Q(A) differenc e spectrum, several modes can be assigned to ring stretching and bending mo des from the neutral and anion radical states of the quinone acceptor. Thes e results provide insight into the PSII/OEC and demonstrate the utility of FTIR techniques in accessing low-frequency modes in proteins.