Single-sided hydrogen bonding to the quinone cofactor in photosystem I probed by selective ¹³C-labelled naphthoquinones and transient EPR

Hydrogen bonding between the protein and one or both of the two 1,4-quinone carbonyl groups of a benzo- or naphtho-quinone constitutes a significant protein-cofactor interaction in photosynthetic reaction centers. The redistribution of charge and spin density due to a particular H-bonding scheme leaves the largest hyperfine couplings (hfc) at the highest density positions, i.e., the nuclei of the carbonyl groups directly involved in H-bonding. The spin density changes at the ring carbon positions are accessed experimentally via electron paramagnetic resonance-determined hfc tensor elements of selective ¹³C isotope labels in one of the two carbonyl groups. Complete hfc tensor data are presented for each of the ¹³C positions in the functional charge-separated state in reaction centers of photosystem I (PS I) isolated from cyanobacteria. A highly asymmetric H-bonding scheme for the A ₁ quinone binding site due to a single dominant H-bond to one carbonyl group is confirmed. A comparison to other well-studied quinone binding sites of other protein-cofactor systems with more complex H-bonding schemes reveals the uniqueness of the PS I site. The single-sided A₁ quinone site provides an ideal test case for the various sets of density functional theory (DFT) calculations that are currently available. While the overall agreement between experimental and calculated data is quite satisfactory, a significant discrepancy is found for the high-spin-density ¹³C position associated with the H-bonded carbonyl. The dominant hfc component (and spin density) is underestimated in the DFT calculations, not only for the high-asymmetry case in PS I, but also for other quinone binding sites with less asymmetry that result from more complex H-bonding schemes. The consequences and potential relevance of this finding for biological function are discussed.