Pulse electron paramagnetic resonance and hyperfine sublevel correlation spectroscopy have been used to investigate nitrogen coordination of the active site of [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F in its oxidized "ready" state. The obtained N-14 hyperfine (A [+1.32, +1.32, +2.07] MHz) and nuclear quadrupole (e(2)qQ/h 1.9 MHz, eta = 0.37) coupling constants were assigned to the N-epsilon of a highly conserved histidine (His88) by studying a hydrogenase preparation in which the histidines were N-15 labeled. The histidine is hydrogen-bonded via its N-is an element of-H to the nickel-coordinating sulfur of a cysteine (Cys549) that carries an appreciable amount of spin density. Through the hydrogen bond a small fraction of the spin density (similar to 1%) is delocalized onto the histidine ring giving rise to an isotropic N-14 hyperfine coupling constant of about 1.6 MHz. These conclusions are supported by density functional calculations. The measured N-14 quadrupole coupling constants are related to the polarization of the N-is an element of-H bond, and the respective hydrogen bond can be classified as being weak.