Richard Cammack

Properties and reactivation of two different deactivated forms of Desulfovibrio gigas hydrogenase

Abstract It has previously been shown that Desulfovibrio gigas hydrogenase, as isolated, has a relatively low activity in the hydrogen-methyl viologen reductase assay, and that the activity is slowly stimulated up to 10-fold by hydrogen or strong reductants. The enzyme, before reductive activation, is also totally inactive in hydrogentritium exchange and hydrogen-dichloroindophenol (DCIP) reductase assays. The behaviour of the enzyme in various states of activation is discussed in terms of three different states: the active state, which is active in all assays, the unready state, which is totally inactive, and the ready state, which does not react with hydrogen, but which is rapidly activated by strong reductants. The conditions for the slow activation of the unready state of D. gigas hydrogenase have been investigated. The rate of activation was independent of enzyme concentration over a wide range, which rules out mechanisms involving intermolecular electron exchange. The rate was only slightly affected by pH in the range 6–9, but was strongly temperature-dependent, with activation energy 88 kJ · mol−1. The enzyme could be activated by dithiothreitol + the mediator dye, Indigo tetrasulphonate, but not by dithiothreitol alone. No effects were seen during treatments with weaker reductants, thioredoxin, iron, sulphide or nickel. These results indicate that the activation does not involve conversions of a metal centre or the cleavage of an accessible disulphide bridge. Presumably it involves an intramolecular change, possibly in the redox state or coordination of a metal centre. The active form of D. gigas hydrogenase was rapidly inactivated by oxygen, producing mostly the unready state, which could be reactivated only slowly. By contrast, anaerobic reoxidation by DCIP was able to convert the enzyme mostly to the ready state. This was identified as being inactive in hydrogen-tritium exchange and hydrogen-DCIP reductase assays but rapidly activated in the hydrogen-methyl viologen reductase assay (DCIP prevents this). It is suggested that a similar oxidation of the active enzyme may take place in the cell as a protection against oxygen.

Electron spin relaxation of iron-sulphur proteins studied by microwave power saturation.

The electron-spin relaxation of iron-sulphur centres in a range of simple proteins (ferredoxin, high-potential iron-sulphur protein and rubredoxin) was investigated by means of the temperature dependence and microwave power saturation of the EPR signal. The proteins containing [2Fe-2S] centres all showed temperature optima higher than those for [4Fe-4S] centres, but the difference between the slowest-relaxing [4Fe-4S] protein (Chromatium high-potential iron-sulphur protein) and the fastest-relaxing [2Fe-2S] protein (Halobacterium halobium ferredoxin) was small. A greater distinction was seen in the power saturation behaviour at low temperature (10--20 K). The behaviour of the signal intensity as a function of microwave power was analyzed in terms of the power for half saturation P 1/2 and the degree of homogeneous/inhomogeneous broadening. The effect of distorting the protein structure by salts, organic solvents and urea was to decrease the electron-spin relaxation rate as shown by a decreased value of P 1/2. The addition of Ni2+ as a paramagnetic perturbing agent caused an increase in the electron-spin relaxation rate of all the proteins, with the exception of adrenal ferredoxin, as shown by an increased P 1/2 and, in a few cases, broadening of the linewidth. Ferricyanide, a commonly used oxidizing agent, has similar effects. These results are discussed in relation to the use of paramagnetic probes to determine whether iron-sulphur centres are near to a membrane surface. Spin-spin interactions between two paramagnetic centres in a protein molecule such as a 2[4Fe-4S] ferredoxin, lead to more rapid electron-spin relaxation. This method was used to detect a spin-spin interaction between molybdenum V and centre Fe-SI in xanthine oxidase.

Redox properties of the ESR‐detectable nickel in hydrogenase from Desulfovibrio gigas

Hydrogenases (EC 1.12) from numerous bacterial and algal sources are known to be iron-sulphur proteins [1]. A number of them either contain nickel [2] or require nickel for activity [3-6]. An electron-spin resonance (ESR) signal, with rhombic symmetry, has been observed in membranes of Methanobacterium bryantii [7]. The signal was tentatively assigned as low-spin Ni(III) because its g-values are all above g = 2, indicating a d 7 system, and there is no hyperfine structure [7]. This assignment was confirmed in a nickel-containing hydrogenase isolated from M. thermoautotrophicum [8] by substitution with 61Ni and observing the expected hyperfine splitting in the spectrum. The ESR spectrum therefore provides a spectroscopic probe for the properties and function of nickel in hydrogenase. Hydrogenase of Desulfovibrio gigas contains 12 atoms of iron and 12 of acid-labile sulphide in a molecule o fM r 89 500 [9]. We have now detected ~1 nickel/atom enzyme molecule. The ESR spectrum of the oxidized protein contains an intense narrow signal at g = 2.02 due to an oxidized iron-sulphur cluster [10]. We have now detected a rhombic signal which is very similar to that from nickel in M. thermoautotrophicum hydrogenase. Though broader, this signal is of significant intensity. By using redox titrations we have measured the midpoint reduction potential, E m of the ESR-detectable nickel, to be -145 mV at pH 7.2. This is higher than the I-I+/H2 couple but much lower than the E m-values normally found for Ni(III) complexes. Furthermore, the E m is pH-dependent, indicating that proton transfer takes place during reduction. The Ni(III) signal indicates no superhyperfme splittings due to interaction with nitrogenous ligands or exchangeable protons. Powe~ saturation studies indicate that the ESR-detectable nickel is distant from the ESR-detectable iron-sulphur cluster. The possibility that the nickel may be involved in the reaction cycle of the enzyme is considered.

The iron-sulphur centres of soluble hydrogenase from Alcaligenes eutrophus

The soluble hydrogenase (hydrogen:NAD+ oxidoreductase (EC 1.12.1.2) from Alcaligenes eutrophus has been purified to homogeneity by an improved procedure, which includes preparative electrophoresis as final step. The specific activity of 57 mumol H2 oxidized/min per mg protein was achieved and the yield of pure enzyme from 200 g cells (wet weight) was about 16 mg/purification. After removal of non-functional iron, analysis of iron and acid-labile sulphur yielded average values of 11.5 and 12.9 atoms/molecule of enzyme, respectively. p-Chloromercuribenzoate was a strong inhibitor of hydrogenase and apparently competed with NAD not with H2. Chelating agents, CO and O2 failed to inhibit enzyme activity. The oxidized hydrogenase showed an EPR spectrum with a small signal at g = 2.02. On reduction the appearance of a high temperature (50--77 K) signal at g = 2.04, 1.95 and a more complex low temperature (less than 30 K) spectrum at g = 2.04, 2.0, 1.95, 1.93, 1.86 was observed. The pronounced temperature dependence and characteristic lineshape of the signals obtained with hydrogenase in 80--85% dimethylsulphoxide demonstrated that iron-sulphur centres of both the [2Fe-2S] and [4Fe-4S] types are present in the enzyme. Quantitation of the EPR signals indicated the existence of two identical centres each of the [4Fe-4S] and of the [2Fe-2S] type. The midpoint redox potentials of the [4Fe-4S] and the [2Fe-2S] centres were determined to be -445 mV and -325 mV, respectively. Spin coupling between two centres, indicated by the split feature of the low temperature spectrum of the native hydrogenase around g = 1.95, 1.93, has been established by power saturation studies. On reduction of the [Fe-4S] centres, the electron spin relaxation rate of the [2Fe-2S] centres was considerably increased. Treatment of hydrogenase with CO caused no change in EPR spectra.

Nickel and iron-sulphur centres in Desulfovibrio gigas hydrogenase: ESR spectra, redox properties and interactions

Abstract Some properties of a nickel species (Niue5f8C) in Desulfovibrio gigas hydrogenase (ferredoxin:H+ oxidoreductase, EC 1.18.99.1), which is associated with the activated state, are described. At temperatures above 20 K this species yields an ESR spectrum at g = 2.19, 2.16, 2.01, but at lower temperatures the spectrum changed into a fast-relaxing species with a complex lineshape. Substitution of the enzyme with 61Ni shows that the complex spectrum is associated with nickel. The splitting was correlated with the presence of a broad ESR signal which probably originates from the reduced [4Fe-4S] clusters. It did not correlated with the reduced [3Fe-xS] cluster. These results indicate that the complex spectrum is due to splitting of the Niue5f8C spectrum by spin-spin interaction with the [4Fe-4S] cluster or clusters. The Niue5f8C species was light-sensitive in frozen samples, and underwent a change in spectrum which was reversed in the dark at temperatures above 200 K. Treatment of the enzyme with carbon monoxide in the presence of hydrogen induced a new type of ESR signal, which disappeared on removal of either hydrogen or carbon monoxide. The Niue5f8C species represents an intermediate oxidation state of the enzyme. The midpoint redox potentials, estimated by mediator titrations under controlled hydrogen/argon gas mixtures, were shown to be strongly pH-dependent. The values at pH 7.0 were estimated to be −270 mV (−120 mV/pH unit) for the appearance of the Niue5f8C ESR signal and −390 mV (−60 mV/pH unit) for its disappearance. The midpoint potential of the broad ESR signal was estimated to be −350 (−60 mV/pH unit). Possible schemes for redox states of the various nickel species are discussed.

ESR-detectable nickel and iron-sulphur centres in relation to the reversible activation of Desulfovibrio gigas hydrogenase

Abstract The electron spin resonance (ESR) spectra of hydrogenase from Desulfovibrio gigas were observed during the activation of the enzyme in the oxidized, ‘unready’, state by hydrogen. Signals from nickel(III) (Ni-A), and the [3Fe-xS] cluster were reduced within less than 5 min, and a broad ESR signal appeared at the same time. On the basis of simultaneous changes in optical absorption spectrum, it is proposed that the broad ESR signal represents one or possibly both [4Fe-4S] clusters in the reduced state. The increase of enzyme activity was much slower (at 20°C), and was accompanied by the appearance of another type of nickel signal (Ni-C), and a further small decrease and the Ni-C signal became more intense. On further reoxidation by the dye dichlorophenolindophenol at pH above 7.0 the enzyme was converted to the ‘ready’ state, which could now be reactivated much more rapidly by strong reductants. The proportion of the ready state correlated with a third type of nickel signal, Ni-B. The unready enzyme could also be slowly activated by milder reducing conditions which reduced Ni-A and the [3Fe-xS] cluster but did not induce significant amounts of the Ni-C and [4Fe-4S]1+ signals. The optical absorption changes indicate that the Ni-A is not coupled to an iron-sulphur cluster. It is proposed that the activation of the enzyme involves reduction of the nickel and possibly iron-sulphur centres, followed by a conformational change which alters the coordination state of nickel, and that the unready state contains Ni(III) in the inactive conformation, the ready state Ni(III) in the active conformation, and the active state Ni(I).

ESR studies of protein A of the soluble methane monooxygenase from Methylococcus capsulatus (Bath)

Abstract ESR spectroscopy of protein A, proposed to be the oxygenase component of the methane monooxygenase (methane, NAD(P)H:oxygen oxidoreductase (hydroxylating), EC 1.14.13.25) from Methylococcus capsulatus (Bath), revealed a minor signal at g = 4.3, and a free radical signal at g = 2.01 in the oxidised state. Upon reduction with sodium dithionite to potentials around 0–100 mV a further rhombic signal appeared with g values less than 2. This spectrum was demonstrated to be from an intermediate reduction level, since on further reduction the signal disappeared. The redox properties of the g = 1.95 signal and its temperature dependence are described. The signal was also formed upon reduction of the protein with protein C of the methane monooxygenase complex, which is known to function as an NADH acceptor-reductase protein. The results are interpreted as indicative of a binuclear Fe centre as the redox site in the protein. The ESR spectra are consistent with a centre of the haemerythin type as opposed to an iron-sulphur cluster.

“Super-reduction” of Chromatium high-potential iron-sulphur protein in the presence of dimethyl sulphoxide

Abstract In solutions containing more than 70% dimethyl sulphoxide (DMSO) by volume, the normal reduced state of Chromatium HIPIP can be further reduced by dithionite to give a form with a decreased absorption around 400 nm and an EPR signal similar to those of the reduced ferredoxins. These changes are reversible and the native HIPIP is recovered on removal of DMSO. These observations favour the hypothesis that the four-iron cluster in ferredoxins and HIPIP can exist in three oxidation states.

Low-temperature magnetic circular dichroism evidence for the conversion of four-iron-sulphur clusters in a ferredoxin from Clostridium pasteurianum into three-iron-sulphur clusters

Oxidation of the 8Fe ferredoxin from Clostridium pasteurianum with potassium ferricyanide, followed by purification on Sephadex G-25 and DE-23 cellulose columns, gives a protein with an intense EPR signal at g 2.01. The low-temperature magnetic circular dichroism (MCD) spectra of this species are different from those of the oxidized high-potential iron protein from Chromatium but identical with the spectra of ferredoxin II from Desulphovibrio gigas. On reduction of the ferricyanide-treated ferredoxin with sodium dithionite only a weak EPR signal with g factors of 2.05, 1.94 and 1.89 is obtained. The low-temperature MCD spectra are strongly temperature dependent with a form similar to those of dithionite-reduced D. gigas ferredoxin II. The MCD magnetization curves are dominated by a species with ground-state effective g factors of gi 8.0 and g⊥ 0.0, which are also similar to those determined recently by low-temperature MCD spectroscopy for D. gigas ferredoxin II. The MCD characteristics are quite different from those of dithionite-reduced ferredoxin from Cl. pasteurianum, untreated with ferricyanide. This establishes the close similarity of the iron-sulphur clusters in ferricyanide-treated Cl. pasteurianum ferredoxin and in D. gigas ferredoxin II. The latter is known to contain a single 3Fe centre, similar to that observed in ferredoxin I from Azotobacter vinelandii by X-ray crystallography. Therefore, it is concluded that the [4Fe-4S] clusters of Cl. pasteurianum ferredoxin are converted to 3Fe clusters on oxidation with ferricyanide.

ESR properties of membrane-bound hydrogenases from aerobic hydrogen bacteria

Analysis of the membrane-bound hydrogenase of Alcaligenes eutrophus, strain H16, indicated a content of 7–9 Fe, 7–9 labile sulphide and 0.6–0.7 nickel atoms per molecule of 98 kDa. The protein, as prepared, gave a complex electron-spin resonance (ESR) spectrum in the oxidized state at low temperatures (below 30 K), with average g value greater than 2.0. This was interpreted as being due to the interaction of [3Fe-χS] or [4Fe-4S]3+ cluster and another paramagnetic centre. At higher temperatures a rhombic spectrum, attributred to Ni(III), was observed. On treatment with mercaptoethanol, or reduction to a redox potential below +160 mV, the low-temperature spectrum changed into a narrow signal at g = 2.02, with increased amplitude. The g = 2.02 signal was reduced reversibly with a midpoint potential of 40 ± 30 mV. The complex spectrum could be restored if the enzyme was rapidly reduced and reoxidized, but not under the conditions of the redox titration, when a broader signal appeared instead. The reductive titration was accompanied by a 3–7-fold increase in enzymic activity. On further reduction of A. eutrophus H16 hydrogenase a complex spectrum with average g value less than 2.0 was observed. This indicate the presence of a [4Fe-45]1+ cluster, interacting with either another such cluster or another paramagnetic centre. This cluster or clusters were estimated to have a midpoint potential of −90 ± 30 mV. The ESR spectra of the hydrogenases of A. eutrophus, type strain, and Pseudomonas pseudoflava, in the oxidized and reduced states, were similar to those of the A. eutrophus H16 enzyme, indicating that they have a similar arrangement of redox centres. The characteristic spectra of the reduced e indicate that they represent a new type of membrane-bound hydrogenase.