Vitamin K-1 (VK1) is an important cofactor of the electron-transfer chain in photosystem I (PS 1), referred to as A(1). The special properties of this quinone result from its unique interaction(s) with the protein surrounding. In particular, a single H-bond to neutral A(1) was identified previously in the X-ray crystal structure of PS I. During light-induced electron transfer in PS 1, A(1), is transiently reduced to the radical anion A(1)(center dot-). In this work, we characterized the electron spin density distribution of A(1)(center dot-) with the aim of understanding the influence of the protein surrounding on it. We studied the light-induced spin-polarized radical pair P(700)(center dot+)A(1)(center dot-) and the photoaccumulated radical anion A(1)(center dot-), using advanced pulse EPR, ENDOR, and TRIPLE techniques at Q-band (34 GHz). Exchange with fully deuterated quinone in the A(1) binding site allowed differentiation between proton hyperfine couplings from the quinone and from the protein surrounding. In addition, DFT calculations on a model of the A(1) site were performed and provided proton hyperfine couplings that were in close agreement with the ones determined experimentally. This combined approach allowed the assignment of proton hyperfine coupling tensors to molecular positions, thereby yielding a picture of the spin density distribution in A(1)(center dot-). Comparison with VK1 center dot- in organic solvents (Epel et al. J. Phys. Chem. B 2006, 110, 11549.) leads to the conclusion that the single H-bond present in both the radical pair P(700)(center dot+)A(1)(center dot-) and the photoaccumulated radical anion A(1)(center dot-) is, indeed, the crucial factor that governs the electronic structure of A(1)(center dot-).