A NEW NONHEME IRON ENVIRONMENT IN PARACOCCUS-DENITRIFICANS ADENYLATE KINASE STUDIED BY ELECTRON-PARAMAGNETIC-RESONANCE AND ELECTRON-SPIN ECHO ENVELOPE MODULATION SPECTROSCOPY

Adenylate kinase from the Gram-negative bacterium Paracoccus denitrifi cans (AK(den)) has structural features highly similar to those of the enzyme from Gram-positive organisms. Atomic absorption spectroscopy of the recombinant protein, which is a dimer, revealed the presence of t wo metals, zinc and iron, each binding most probably to one monomer. U nder oxidizing conditions, the electron paramagnetic resonance (EPR) s pectrum of AK(den) at 4.2 K consists of features at g = 9.23, 4.34, 4. 21, and 3.68. These features are absent in the ascorbate-reduced prote in and are characteristic of a S = 5/2 spin system in a rhombic enviro nment with E/D = 0.24 and are assigned to a non-heme Fe3+ (S = 5/2) ce nter. The zero-field splitting parameter D (D = 1.4 +/- 0.2 cm(-1)) wa s estimated from the temperature dependence of the EPR spectra. These EPR characteristic as well as the difference absorption spectrum (oxid ized minus reduced) of AK(den) are similar to those reported for the n on heme iron protein rubredoxin. Nevertheless, the redox potential of the Fe2+/Fe3+ couple in AK(den) was measured at +230 +/- 30 mV, which is more positive than the redox potential of the non-heme iron in rubr edoxin. Binding of cyanide converts the iron from the high-spin (S = 5 /2) to the low-spin (S = 1/2) spin state. The EPR spectrum of the non- heme Fe3+(S = 1/2) in the presence of cyanide has g values of 2.45, 2. 18, and 1.92 and spin-Hamiltonian parameters R/lambda = 7.4 and R/mu = 0.56. The conversion of the non-heme iron to the low-spin (S = 1/2) s tate allowed the study of its local environment by electron spin echo envelope modulation spectroscopy (ESEEM). The ESEEM data revealed the existence of N-14 or N-15 nuclei coupled to the low-spin iron after ad dition of (KCN)-N-14 or (KCN)-N-15 respectively. This demonstrated tha t iron in AK(den) has at least one labile coordination position that c an be easily occupied by cyanide. Other possible magnetic interactions with nitrogen(s) from the protein are discussed.