Reduced inorganic sulfur compounds are utilized by many bacteria as electron donors to photosynthetic or respiratory electron transport chains. This metabolism is a key component of the biogeochemical sulfur cycle. The SoxAX protein is a heterodimeric c ‐type cytochrome involved in thiosulfate oxidation. The crystal structures of SoxAX from the photosynthetic bacterium Rhodovulum sulfidophilum have been solved at 1.75 Å resolution in the oxidized state and at 1.5 Å resolution in the dithionite‐reduced state, providing the first structural insights into the enzymatic oxidation of thiosulfate. The SoxAX active site contains a haem with unprecedented cysteine persulfide (cysteine sulfane) coordination. This unusual post‐translational modification is also seen in sulfurtransferases such as rhodanese. Intriguingly, this enzyme shares further active site characteristics with SoxAX such as an adjacent conserved arginine residue and a strongly positive electrostatic potential. These similarities have allowed us to suggest a catalytic mechanism for enzymatic thiosulfate oxidation. The atomic coordinates and experimental structure factors have been deposited in the PDB with the accession codes 1H31, 1H32 and 1H33.

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5′-phosphate-dependent enzyme responsible for the degradation of the inhibitory neurotransmitter GABA. GABA-AT is a validated target for antiepilepsy drugs because its selective inhibition raises GABA concentrations in brain. The antiepilepsy drug, γ-vinyl-GABA (vigabatrin) has been investigated in the past by various biochemical methods and resulted in several proposals for its mechanisms of inactivation. In this study we solved and compared the crystal structures of pig liver GABA-AT in its native form (to 2.3-Å resolution) and in complex with vigabatrin as well as with the close analogue γ-ethynyl-GABA (to 2.3 and 2.8 Å, respectively). Both inactivators form a covalent ternary adduct with the active site Lys-329 and the pyridoxal 5′-phosphate (PLP) cofactor. The crystal structures provide direct support for specific inactivation mechanisms proposed earlier on the basis of radio-labeling experiments. The reactivity of GABA-AT crystals with the two GABA analogues was also investigated by polarized absorption microspectrophotometry. The spectral data are discussed in relation to the proposed mechanism. Intriguingly, all three structures revealed a [2Fe-2S] cluster of yet unknown function at the center of the dimeric molecule in the vicinity of the PLP cofactors.

The bacterial Sox (sulfur oxidizing) system allows the utilization of inorganic sulfur compounds in energy metabolism. Central to this process is the SoxYZ complex that carries the pathway intermediates on a cysteine residue near the C terminus of SoxY. Crystal structures have been determined for Paracoccus pantotrophus SoxYZ with the carrier cysteine in the underivatized state, conjugated to the polysulfide mimic β-mercaptoethanol, and as the sulfonate adduct pathway intermediate. The carrier cysteine is located on a peptide swinging arm and is bracketed on either side by diglycine dipeptides acting as molecular universal joints. This structure provides a novel solution to the requirement that the cysteine-bound intermediates be able to access and orient themselves within the active sites of multiple partner enzymes. Adjacent to the swinging arm there is a conserved, deep, apolar pocket into which the β-mercaptoethanol adduct extends. This pocket would be well suited to a role in protecting labile pathway intermediates from adventitious reactions.

The microorganisms that evolved at low temperatures express cold-adapted enzymes endowed with unique catalytic properties in comparison to their mesophilic homologues, i.e., higher catalytic efficiency, improved flexibility, and lower thermal stability. Cold environments are therefore an attractive research area for the discovery of enzymes to be used for investigational and industrial applications in which such properties are desirable. In this work, we will review the literature on cold-adapted enzymes specifically focusing on those discovered in the bioprospecting of polar marine environments, so far largely neglected because of their limited accessibility. We will discuss their existing or proposed biotechnological applications within the framework of the more general applications of cold-adapted enzymes.

To understand why the classical two-state allosteric model of Monod, Wyman, and Changeux explains cooperative oxygen binding by hemoglobin but does not explain changes in oxygen affinity by allosteric inhibitors, we have investigated the kinetic properties of unstable conformations transiently trapped by encapsulation in silica gels. Conformational trapping reveals that after nanosecond photodissociation of carbon monoxide a large fraction of the subunits of theTquaternary structure has kinetic properties almost identical to those of subunits of theRquaternary structure. Addition of allosteric inhibitors reduces both the fraction ofR-like subunits and the oxygen affinity of theTquaternary structure. These kinetic and equilibrium results are readily explained by a recently proposed generalization of the Monod-Wyman-Changeux model in which a preequilibrium between two functionally different tertiary, rather than quaternary, conformations plays the central role.

To understand the interplay between tertiary and quaternary transitions associated with hemoglobin function and regulation, oxygen binding curves were obtained for hemoglobin A fixed in the T quaternary state by encapsulation in wet porous silica gels. At pH 7.0 and 15°C, the oxygen pressure at half saturation (p50) was measured to be 12.4 ± 0.2 and 139 ± 4 torr for hemoglobin gels prepared in the absence and presence of the strong allosteric effectors inositol hexaphosphate and bezafibrate, respectively. Both values are in excellent agreement with those found for the binding of the first oxygen to hemoglobin in solution under similar experimental conditions. The corresponding Hill coefficients of hemoglobin gels were 0.94 ± 0.02 and 0.93 ± 0.03, indicating, in the frame of the Monod, Wyman, and Changeux model, that high and low oxygen‐affinity tertiary T‐state conformations have been isolated in a pure form. The values, slightly lower than unity, reflect the different oxygen affinity of α‐ and β‐hemes. Significantly, hemoglobin encapsulated in the presence of the weak effector phosphate led to gels that show intermediate oxygen affinity and Hill coefficients of 0.7 to 0.8. The heterogeneous oxygen binding results from the presence of a mixture of the high and low oxygen‐affinity T states. The Bohr effect was measured for hemoglobin gels containing the pure conformations and found to be more pronounced for the high‐affinity T state and almost absent for the low‐affinity T state. These findings indicate that the functional properties of the T quaternary state result from the contribution of two distinct, interconverting conformations, characterized by a 10‐fold difference in oxygen affinity and a different extent of tertiary Bohr effect. The very small degree of T‐state cooperativity observed in solution and in the crystalline state might arise from a ligand‐induced perturbation of the distribution between the high‐ and low‐affinity T‐state conformations.