Solution structure of Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c 3 : structural basis for functional cooperativity 1 1Edited by P. E. Wright

Abstract

Desulfovibrio vulgaris cytochrome c 3 is a 14 kDa tetrahaem cytochrome that plays a central role in energy transduction. The three-dimensional structure of the ferrocytochrome at pH 8.5 was solved through twodimensional 1 H-NMR. The structures were calculated using a large amount of experimental information, which includes upper and lower distance limits as well as dihedral angle restraints. The analysis allows for fast-¯ipping aromatic residues and¯exibility in the haem plane. The structure was determined using 2289 upper and 2390 lower distance limits, 63 restricted ranges for the f torsion angle, 88 stereospeci®c assignments out of the 118 stereopairs with non-degenerate chemical shifts (74.6%), and 115 out of the 184 nuclear Overhauser effects to fastipping aromatic residues (62.5%), which were pseudo-stereospeci®cally assigned to one or the other side of the ring. The calculated NMR structures are very well de®ned, with an average root-mean-square deviation value relative to the mean coordinates of 0.35 A Ê for the backbone atoms and 0.70 A Ê for all heavy-atoms. Comparison of the NMR structures of the ferrocytochrome at pH 8.5 with the available X-ray structure of the ferricytochrome at pH 5.5 reveals that the general fold of the molecule is very similar, but that there are some distinct differences. Calculation of ring current shifts for the residues with signi®cantly different conformations con®rms that the NMR structures represent better its solution structure in the reduced form. Some of the localised differences, such as a reorientation of Thr24, are thought to be state-dependent changes that involve alterations in hydrogen bond networks. An important rearrangement in the vicinity of the propionate groups of haem I and involving the covalent linkage of haem II suggests that this is the critical region for the functional cooperativities of this protein. # 1998 Academic PressKeywords: multihaem cytochrome; NMR structure; ring current shifts; redox-Bohr effect; cooperativity *Corresponding author IntroductionThe functional cooperativity between different regions of certain proteins is a fundamental property for controlling and coordinating important chemical events in the living cell. Although the molecular basis for the ®ne regulation of several types of cooperativity mechanisms has been E-mail address of the corresponding author: xavier@itqb.unl.pt Abbreviations used: DvHc 3 , Desulfovibrio vulgaris (Hildenborough) cytochrome c 3 ; NMR, nuclear magnetic resonance; 2D, two-dimensional; 2D-1 H-NMR, two-dimensional proton NMR; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser spectroscopy; COSY, correlation spectroscopy; DQF-COSY, double-quantum ®ltered COSY; TOCSY, total correlation spectroscopy; TPPI, time-proportional phase incrementation; ppm, parts per million; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; MD, molecular dynamics; RMSD, root-mean-square deviation; lol, lower distance limit; upl, upper distance limit.0022±2836/98/340719±21 $30.00/0 # 1998 Academic Press successfully established ...

Conformational Component in the Coupled Transfer of Multiple Electrons and Protons in a Monomeric Tetraheme Cytochrome

“…The detailed thermodynamic properties that favor a coupled two-electron step modulated by a redox-Bohr effect in Ddc3, i.e. a proton-assisted two-electron step, are different from those reported for other structurally homologous cytochromes c 3 (2,3,24,32). Thus, the study of these various proteins yields information on the variety of strategies that Nature can employ to achieve a common function using the same structural arrangement of the redox active sites.…”

“…The structural analysis of bacteriorhodopsin trapped in the M photointermediate state (1), the structures in the oxidized and reduced forms of cytochromes c 3 that perform a coupled two-electron step associated with proton transfer (2,3), and the establishment of the coupled transfer of electrons and protons to the 3Fe-4S cluster of Azotobacter vinelandii ferredoxin (4) are just a few recent examples where results from different techniques are integrated in a description at the atomic level of the energy-transducing events. The phenomenon of energy transduction relies on coupled events (5), whether they involve only electrostatic interactions or structural rearrangements of the active sites or its surroundings (mechano-chemical coupling), which may be more important than the electrostatic component of the overall coupling (6).…”

Effect of Hydrogen-Bond Networks in Controlling Reduction Potentials in Desulfovibrio vulgaris (Hildenborough) Cytochrome c3 Probed by Site-Specific Mutagenesis

“…This is consistent with a relative destabilization of the oxidized state of the protein since the replacement of T24 by valine prevents the formation of a network of hydrogen bonds that stabilizes the oxidized state of the wild type (28).…”

“…In T24VDVHc 3 , differences caused near heme III by the mutation could be responsible for this small variation, through the modulation of the dielectric constant or movement of the ionizable group. Previous work showed that the redox-Bohr effect in DVHc 3 is controlled by heme I propionates and neighboring residues (11,27,28), and since the residue mutated is not located close to heme I, the four macroscopic reduction potentials have a similar pH dependence in both cytochromes, although it is shifted to significantly higher values in T24VDVHc 3 ( Figure 5).…”

Proton-assisted Two-electron Transfer in Natural Variants of Tetraheme Cytochromes from Desulfomicrobium Sp.

“…Recent studies allowed the identification of the key residues involved in homotropic and/or heterotropic cooperativities in type I cytochromes c 3 from Desulfovibrio gigas (Dg) (9) and Desulfovibrio vulgaris Hildenborough (DvH) (10,11). Site-directed mutagenesis studies were used to further probe the role of some specific residues in the modulation of the redox properties of these proteins (12)(13)(14).…”