RESUMEN
The O(2)-tolerant [NiFe] hydrogenases of Ralstonia eutropha are capable of H(2) conversion in the presence of ambient O(2). Oxygen represents not only a challenge for catalysis but also for the complex assembling process of the [NiFe] active site. Apart from nickel and iron, the catalytic center contains unusual diatomic ligands, namely two cyanides (CN(-)) and one carbon monoxide (CO), which are coordinated to the iron. One of the open questions of the maturation process concerns the origin and biosynthesis of the CO group. Isotope labeling in combination with infrared spectroscopy revealed that externally supplied gaseous (13)CO serves as precursor of the carbonyl group of the regulatory [NiFe] hydrogenase in R. eutropha. Corresponding (13)CO titration experiments showed that a concentration 130-fold higher than ambient CO (0.1 ppmv) caused a 50% labeling of the carbonyl ligand in the [NiFe] hydrogenase, leading to the conclusion that the carbonyl ligand originates from an intracellular metabolite. A novel setup allowed us to the study effects of CO depletion on maturation in vivo. Upon induction of CO depletion by addition of the CO scavenger PdCl(2), cells cultivated on H(2), CO(2), and O(2) showed severe growth retardation at low cell concentrations, which was on the basis of partially arrested hydrogenase maturation, leading to reduced hydrogenase activity. This suggests gaseous CO as a metabolic precursor under these conditions. The addition of PdCl(2) to cells cultivated heterotrophically on organic substrates had no effect on hydrogenase maturation. These results indicate at least two different pathways for biosynthesis of the CO ligand of [NiFe] hydrogenase.
Asunto(s)
Proteínas Bacterianas/química , Monóxido de Carbono/química , Cupriavidus necator/enzimología , Hidrogenasas/química , Dominio Catalítico , Ligandos , Paladio/químicaRESUMEN
Most of the biochemical and biophysical processes of proteins take place at membranes, and are thus under the influence of strong local electric fields, which are likely to affect the structure as well as the reaction mechanism and dynamics. To analyse such electric field effects, biomimetic interfaces may be employed that consist of membrane models deposited on nanostructured metal electrodes. For such devices, surface-enhanced resonance Raman and IR absorption spectroscopy are powerful techniques to disentangle the complex interfacial processes of proteins in terms of rotational diffusion, electron transfer, and protein and cofactor structural changes. The present article reviews the results obtained for the haem protein cytochrome c, which is widely used as a model protein for studying the various reaction steps of interfacial redox processes in general. In addition, it is shown that electric field effects may be functional for the natural redox processes of cytochrome c in the respiratory chain, as well as for the switch from the redox to the peroxidase function, one of the key events preceding apoptosis.
Asunto(s)
Biomimética , Citocromos c/química , Citocromos c/metabolismo , Proteínas/metabolismo , Análisis Espectral/métodos , Electrones , Sondas Moleculares , Estructura Molecular , Oxidación-Reducción , Unión Proteica , VibraciónRESUMEN
The catalytic cycle of the anaerobic [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F (DvMF) both in solution and immobilized on an Au electrode was studied by IR spectroscopic and electrochemical methods. IR spectroelectrochemistry in solution at different pH values allows the identification of the various redox-states of the active site and the determination of the midpoint potentials, as well as their acid-base equilibria. The spectroscopic characterization was based on the unique marker bands of the CN and CO stretching modes of the Ni-Fe center and served as reference for the surface-enhanced IR absorption (SEIRA) study of the immobilized enzyme. Using structural models of hydrogenases from DvMF and Desulfovibrio gigas , dipole moment calculations were carried out to guide the immobilization strategy. In view of the high dipole moment of about 1100 D pointing through the negatively charged area surrounding the distal [FeS] cluster, the Au electrode was coated by a self-assembled monolayer of amino-terminated mercaptanes which, due to the positively charged head groups, permit a durable electrostatic binding of the protein. SEIRA spectroscopy revealed a structurally and functionally intact active site as demonstrated by the reversible activation and inactivation under hydrogen and argon, respectively. Cyclic voltammetry on the immobilized enzyme demonstrate a reversible anaerobic inactivation upon changing the applied potential. The "switch" potential (E(switch)) associated with the reductive reactivation was determined to be -33 mV (vs normal hydrogen electrode). However, the catalytic current decreased on the time scale of hours during continuous cycling. SEIRA experiments demonstrate that the loss of catalytic activity is not due to protein desorption but is rather related to a slow degradation of the active site, possibly initiated by the attack of reactive species electrochemically generated from residual traces of oxygen in solution.
Asunto(s)
Desulfovibrio vulgaris/enzimología , Técnicas Electroquímicas , Oro/química , Hidrogenasas/química , Espectrofotometría Infrarroja , Argón/química , Dominio Catalítico , Electrodos , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Hidrógeno/química , Hidrogenasas/metabolismo , Oxidación-ReducciónRESUMEN
[NiFe] hydrogenases catalyze the reversible conversion of H2 into protons and electrons. The reaction takes place at the active site, which is composed of a nickel and an iron atom and three diatomic ligands, two cyanides and one carbon monoxide, bound to the iron. The NiFe(CN-)2CO cofactor is synthesized by an intricate posttranslational maturation process, which is mediated by a set of six conserved Hyp proteins. Depending on the cellular location and the physiological function, additional auxiliary proteins are involved in hydrogenase biosynthesis. Here we present evidence that the auxiliary proteins HoxL and HoxV assist in assembly of the Fe(CN-)2CO moiety. This unit was identified as a cofactor intermediate of the oxygen-tolerant membrane-bound [NiFe] hydrogenase (MBH) in the beta-proteobacterium Ralstonia eutropha H16. Both HoxL and HoxV proved to be essential for H2-oxidizing activity and MBH-driven growth on H2. Copurification studies revealed that HoxL and HoxV directly interact with the hydrogenase apoprotein. HoxV forms complexes with HoxL and HypC, a HoxL paralogue that is essential for cofactor assembly. These observations suggest that HoxL acts as a specific chaperone assisting the transfer of the Fe(CN-)2CO cofactor intermediate from the Hyp machinery to the MBH. This shuttle also involves the scaffold protein HoxV. Indeed, infrared spectroscopy and metal analysis identified for the first time a non-redox-active Fe(CN-)2CO intermediate coordinated to HoxV.
Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/enzimología , Hidrogenasas/biosíntesis , Hidrogenasas/química , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Dominio Catalítico , Secuencia Conservada , Hidrogenasas/genética , Hidrogenasas/aislamiento & purificación , Datos de Secuencia Molecular , Unión Proteica , Ralstonia/enzimología , Ralstonia/genética , Alineación de Secuencia , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Time-resolved surface enhanced infrared absorption (SEIRA) spectroscopy is employed to analyse the dynamics of the protein structural changes coupled to the electron transfer process of immobilised cytochrome c (Cyt-c). Upon electrostatic binding of Cyt-c to Au electrodes coated with self-assembled monolayers (SAMs) of carboxyl-terminated thiols, cyclic voltammetric measurements demonstrate a reversible redox process with a redox potential that is similar to that of Cyt-c in solution, and a non-exponential distance-dependence of the electron transfer rate as observed previously (D. H. Murgida and P. Hildebrandt, Chem. Soc. Rev. 2008, 37, 937). On the basis of characteristic redox-state-sensitive amide I bands, the protein structural changes triggered by the electron transfer are monitored by rapid scan and step scan SEIRA spectroscopy in combination with the potential jump technique. Whereas the temporal evolution of the conjugate bands at 1693 and 1673 cm(-1) displays the same rate constants as electron transfer, the time-dependent changes of the 1660-cm(-1) band are slower by about a factor of 2. The study demonstrates that time-resolved SEIRA spectroscopy provides further information about the dynamics and mechanism of interfacial processes of redox proteins, thereby complementing the results obtained from other surface-sensitive techniques. In comparison with previous surface enhanced resonance Raman spectroscopic findings, the present results are discussed in terms of the local electric field strengths at the Au/SAM/Cyt-c interface.