Pulsed proton-deuterium ^{1, 2}H ENDOR and ²H-²H TRIPLE resonance of H-bonds and cysteinyl β-CH₂ of the D. gigas hydrogenase [3Fe-4S]⁺ cluster

This paper presents the results of ²H Mims and ¹H Davies pulsed ENDOR of the [3Fe-4S]⁺ cluster of Desulfovibrio gigas hydrogenase. It also describes the first ²H Mims pulsed TRIPLE-resonance experiments, on the exchangeable deuterons. The ¹H ENDOR data show that the intrinsic isotropic hyperfine couplings for the β-CH₂protons of the three cysteines bound to iron fall in the range 1.3 ≲ a ≲ 1.9 MHz. These values, which often had been estimated at a = 1 MHz, will allow better interpretation of solution NMR data for paramagnetic [nFe-wS] clusters. The spectra show well-resolved signals from three strong NH…S H-bonds to the cluster. Mims ²H-²H TRIPLE-resonance has been used to assign the exchangeable deuterons and to obtain relative signs of the ^{1, 2}H hyperfine couplings. Analysis of the hyperfine interactions in terms of spin-coupling among the three Fe(III) ions of the cluster, along with examination of the X-ray structures of several [3Fe-4S]⁺ clusters, gives an intrinsic hyperfine coupling of a(₁H) ~ 3-4 MHz for each of the three strong H-bonds and actually allows us to propose the distribution of H-bonds among the seven sulfur atoms within the [Fe₃S⁴(Cys)₃]^2- center.