Thermodynamic and kinetic characterization of protonation exchanges in GFPs: a way to mutants with tailored optical properties (original) (raw)
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Biochemical Journal, 2009
Multihaem cytochromes that could form protein “nanowires” were identified in the Geobacter sulfurreducens genome, and represent a new type of multihaem cytochrome. The sequences of these proteins, two with 12 haems (GSU1996, GSU0592) and one with 27 haems (GSU2210), suggest that they are formed with domains homologous to the trihaem cytochrome c7. Although all three haems have bis-His co-ordination in cytochromes c7, in each domain of the above polymers, the haem equivalent to haem IV has His-Met co-ordination. We previously determined the structure and measured the macroscopic redox potential of one representative domain (domain C) of a dodecahaem cytochrome (GSU1996). In the present study, the microscopic redox properties of the individual haem groups of domain C were determined using NMR and UV–visible spectroscopies. The reduction potentials of the haems for the fully reduced and protonated protein are different from each other (haem I, −106 mV; haem III, −136 mV; and haem IV, −...
Journal of Biological Inorganic Chemistry, 2002
NMR and visible spectroscopy were used to characterize the type II tetraheme cytochrome c 3 isolated from the periplasmic space of Desulfovibrio africanus, a sulfate-reducing bacterium. Although structurally similar to other cytochromes c 3, this protein displays distinct functional properties. Proton NMR signals from the four hemes were assigned to the structure in the ferri- and ferrocytochromes using two-dimensional NMR experiments. The thermodynamic parameters of the hemes and of an acid-base center in the type II cytochrome c 3 were determined using NMR and visible spectroscopies. The thermodynamic features indicate that electrostatic effects dominate all of the interactions between the centers and no positive cooperativity between hemes is observed. The redox-Bohr effect in this protein is associated with the acid-base equilibrium of a propionate of heme II instead of propionate 13 of heme I as is the case for all of the type I cytochromes c 3. These novel functional properties are analyzed together with the redox-linked structural differences reported in the literature and reveal a mechanistic basis for type II cytochromes c 3 having a physiological function that is different from that of type I cytochromes c 3.
A Membrane-Bound Cytochromec3: A Type II Cytochromec3 fromDesulfovibrio vulgaris Hildenborough
ChemBioChem, 2001
A new tetraheme cytochrome c3 was isolated from the membranes of Desulfovibrio vulgaris Hildenborough (DvH). This cytochrome has a molecular mass of 13.4 kDa, a pI of 5.5, and contains four hemes c with apparent reduction potentials of-170 mV,-235 mV,-260 mV and-325 mV at pH 7.6. The complete sequence of the new cytochrome, retrieved from the preliminary data of the DvH genome, shows that this cytochrome is homologous to the "acidic" cytochrome c3 from Desulfovibrio africanus (Da). A model for the structure of the DvH cytochrome was built based on the structure of the Da cytochrome. Both cytochromes share structural features that distinguish them from other cytochromes c3, such as a solvent-exposed heme 1 surrounded by an acidic surface area, and a heme 4 which lacks most of the surface lysine patch proposed to be the site of hydrogenase interaction in other cytochromes c3. Furthermore, in contrast to previously discovered cytochromes c3, the genes coding for these two cytochromes are adjacent to genes coding for two membrane associated FeS proteins, which indicates that they may be part of membrane-bound oxido-reductase complexes. Altogether these observations suggest that the DvH and Da cytochromes are a new type of cytochromes c3 (Type II: TpII-c3) with different redox partners and physiological function than the other cytochromes c3 (Type I: TpI-c3). The DvH TpII-c3 is reduced with considerable rates by the two membrane-bound [NiFe] and [NiFeSe] hydrogenases, but catalytic amounts of TpI-c3 increase these rates two and four-fold, respectively. With the periplasmic [Fe] hydrogenase TpII-c3 is reduced much slower than TpI-c3, and no catalytic effect of TpI-c3 is observed.
Structure, 1997
Background: Cytochromes c 4 are dihaem cytochromes c found in a variety of bacteria. They are assumed to take part in the electron-transport systems associated with both aerobic and anaerobic respiration. The cytochrome c 4 proteins are located in the periplasm, predominantly bound to the inner membrane, and are able to transfer electrons between membrane-bound reduction systems and terminal oxidases. Alignment of cytochrome c 4 sequences from three bacteria, Pseudomonas aeruginosa, Pseudomonas stutzeri and Azotobacter vinelandii, suggests that these dihaem proteins are composed of two similar domains. Two distinctly different redox potentials have been measured for the Ps. stutzeri cytochrome c 4 , however. Results: The crystal structure of the dihaem cytochrome c 4 from Ps. stutzeri has been determined to 2.2 Å resolution by isomorphous replacement. The model, consisting of two entire cytochrome c 4 molecules and 138 water molecules in the asymmetric unit, was refined to an R value of 20.1% for all observations in the resolution range 8-2.2 Å. The molecule is organized in two cytochrome c-like domains that are related by a pseudo-twofold axis. The symmetry is virtually perfectly close to the twofold axis, which passes through a short hydrogen bond between the two haem propionic acid groups, connecting the redox centre of each domain. This haem-haem interaction is further stabilized by an extensive symmetrical hydrogen-bond network. The twofold symmetry is not present further away from the axis, however, and the cytochrome c 4 molecule can be considered to be a dipole with charged residues unevenly distributed between the two domains. The haem environments in the two domains show pronounced differences, mainly on the methionine side of the haem group. Conclusions: The structure, in conjunction with sequence alignment, suggests that the cytochrome protein has evolved by duplication of a cytochrome c gene. The difference in charge distribution around each haem group in the two domains allows the haem group in the N-terminal domain to be associated with the lower redox potential of 241 mV and the C-terminal haem group with the higher potential of 328 mV. The molecular dipole characteristic of cytochrome c 4 is important for its interaction with, and recognition of, its redox partners. In cytochrome c 4 , the hydrogen-bond network (between residues that are conserved in all known cytochrome c 4 subspecies) seems to provide an efficient pathway for an intramolecular electron transfer that can ensure cooperativity between the two redox centres. The C-pyrrole corners of the haem edges are potential sites for external electron exchange.
Functional properties of type I and type II cytochromes c 3 from Desulfovibrio africanus
Biochimica Et Biophysica Acta-bioenergetics, 2007
Type I cytochrome c 3 is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c 3 . This work presents the NMR assignment of the haem substituents in type I cytochrome c 3 isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c 3 belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.
Structure, 1999
Background: Haem-containing proteins are directly involved in electron transfer as well as in enzymatic functions. The nine-haem cytochrome c (9Hcc), previously described as having 12 haem groups, was isolated from cells of Desulfovibrio desulfuricans ATCC 27774, grown under both nitrate- and sulphate-respiring conditions.Results: Models for the primary and three-dimensional structures of this cytochrome, containing 292 amino acid residues and nine haem groups, were derived using the multiple wavelength anomalous dispersion phasing method and refined using 1.8 Å diffraction data to an R value of 17.0%. The nine haem groups are arranged into two tetrahaem clusters, with Fe–Fe distances and local protein fold similar to tetrahaem cytochromes c3, while the extra haem is located asymmetrically between the two clusters.Conclusions: This is the first known three-dimensional structure in which multiple copies of a tetrahaem cytochrome c3-like fold are present in the same polypeptide chain. Sequence homology was found between this cytochrome and the C-terminal region (residues 229–514) of the high molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough (DvH Hmc). A new haem arrangement in domains III and IV of DvH Hmc is proposed. Kinetic experiments showed that 9Hcc can be reduced by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774, but that this reduction is faster in the presence of tetrahaem cytochrome c3. As Hmc has never been found in D. desulfuricans ATCC 27774, we propose that 9Hcc replaces it in this organism and is therefore probably involved in electron transfer across the membrane.