UNUSUAL LACK OF INTERNAL MOBILITY AND FAST OVERALL TUMBLING IN OXIDIZED FLAVODOXIN FROM ANACYSTIS-NIDULANS

Anacystis nidulans flavodoxin, an electron-transfer protein containing a flavin mononucleotide (FMN) molecule as its prosthetic group, has a redox potential for the oxidized/semiquinone equilibrium close to tha t of free flavin. Whereas the redox potential for the semiquinone/hydr oquinone equilibrium is more negative. To gain an understanding of the contribution of mobility to redox potential modulation, we studied th e backbone mobility of the oxidized A, nidulans flavodoxin at pH 6.6, 303 K by N-15 NMR relaxation measurements. The spin-lattice relaxation rate constants (R-N(N-z)=1/T-1), spin-spin relaxation rate constants (R-N(N-x,N-y)=1/T-2) and H-1-N-15 nuclear Overhauser effects (NOE) wer e obtained for 143 of the 166 protonated backbone N-15 nuclei and for the FMN N3 nucleus without ambiguity. The N-15 T-1, T-2 and NOE data w ere analyzed by reduced spectral density mapping, and the so-called mo del-free approach. In contrast to most other proteins studied with N-1 5 relaxation experiments, we found an almost complete absence of inter nal mobility. The overall correlation time of this 169-residue flavodo xin (>19 kDa) is significantly shorter (7.4 to 7.8 ns) than that of ot her proteins of this size, suggesting that the absence of internal mob ility is correlated with faster overall rotational diffusion. The unif ormity of the motional parameters along the backbone is in strong cont rast to the crystallographic B-factors, which vary significantly along the sequence in this and other flavodoxins. The NMR relaxation parame ters are primarily sensitive to rotational diffusive motions of the N- H bond vectors, while the crystallographic B-factors would be sensitiv e to translational internal motions as well. However, the large B-fact ors in this protein may originate from crystal packing and crystal lat tice disorder. The relatively fast overall tumbling results in sharp N MR resonances. Hence, much larger proteins with such favorable dynamic behavior could be excellent candidates for studies by NMR. (C) 1997 A cademic Press Limited.