J.W. van der Zwaan

Monovalent nickel in hydrogenase from Chromatium vinosum: Light sensitivity and evidence for direct interaction with hydrogen

Redox titrations with hydrogenase from Chromatium vinosum show that its nickel ion can exist in 3, possibly 4, different redox states: the 3 +, 2 +, 1 + and possibly a zero valent state. The 1 + state is unstable: oxidation to Ni(II) occurs unless H2 gas is present. The Ni(I) coordination, but not that of Ni(III), is highly light sensitive. A photoreaction occurs on illumination. It is irreversible below 77 K, but reversible at 200 K. The rate of this photodissociation reaction in 2H2O is nearly 6‐times slower than in H2O, indicating the breakage of a nickel‐hydrogen bond. This forms the first evidence for an H atom in the direct coordination sphere of Ni in hydrogenase and for the involvement of this metal in the reaction with hydrogen.

Effect of 17O2 and 13CO on EPR espectra of nickel in hydrogenase from Chromatium vinosum

Oxygen, either molecular oxygen or a reduction adduct, can tightly bind in the vicinity of the two forms of trivalent nickel occurring in hydrogenase from Chromatium vinosum, as evident from studies with 17O-enriched O2. This oxygen is not in the first coordination sphere of nickel. As has been reported earlier for hydrogenase from Desulfovibrio gigas (Fernandez, V.M., Hatchikian, A.C., Patil, D.S. and Cammack, R. (1986) Biochim. Biophys. Acta 883, 145-154), also the relative activity of the C.vinosum enzyme correlates well with the presence of only one of the two Ni(III) forms in the oxidized preparation. These results make it less likely that a specific oxygenation of only one of the Ni(III) forms would be the reason for the reversible inactivation of nickel hydrogenases by oxygen. Reaction of H2-reduced enzyme with 13CO now demonstrated beyond doubt that: (i) One 13CO molecule is a direct ligand to nickel in axial position; and (ii) hydrogen binds at the same coordination site as CO. It can also be concluded that hydrogen is not bound as a hydride ion, but presumably as molecular hydrogen. A simple way to explain the EPR spectra from the 13CO-adduct of the enzyme is to assume a monovalent state for the nickel.

Five new EPR signals assigned to nickel in methyl-coenzyme M reductase from Methanobacterium thermoautotrophicum, strain Marburg

Abstract EPR spectroscopy of intact cells and of cell extracts from Methanobacterium thermoautotrophicum, strain Marburg, revealed the existence of five new EPR signals assigned to factor F430 in methyl-coenzyme M reductase, in addition to the one discovered recently in the purified enzyme (Albracht, S.P.J., Ankel-Fuchs, D., Van der Zwaan, J.W., Fontijn, R.D. and Thauer, R.K. (1986) Biochim. Biophys. Acta 870, 50–57), which is further referred to as MCR-ox1. Two of the new signals, a strongly axial one, termed MCR-red1, and a rhombic one, termed MCR-red 2, could be evoked by incubation of intact cells with H2 at 60°C, but not at 4°C. Subsequent incubation under N2 did not change these signals, but they quickly disappeared on anaerobic contact with CO2 at 60°C, but not at 4°C. This behaviour indicates that under physiological conditions nickel in factor F430 of methyl-coenzyme M reductase is redox active and, the the reduced state, can occur in two forms with strongly different ligand fields. The possible significance of these findings for the mechanism of action of methyl-coenzyme M reductase in intact cells is discussed. A third new signal, termed MCR-ox2, was observed after oxidation by air of intact cells previously reduced by H2 at 60°C. In cell extract and isolated methyl-coenzyme M reductase a fourth new signal, called MCR-dark, could be evoked under several conditions. The species responsible for this signal was highly sensitive to light. Illumination below 100 K converted the signal to yet another one of rhombic nature (termed MCR-light) which was similar but not identical to the rhombic signal (MCR-red2) observed in intact cells. At 170 K the change was reversible. From the latter results it is concluded that nickel in factor F430 of methyl-coenzyme M reductase can exist in a form where its coordination can be greatly changed by illumination below 100 K.

EPR Spectrum at 4, 9 and 35 GHz of hydrogenase from Chromatium vinosum. Direct evidence for spin-spin interaction between Ni(III) and the ironsulphur cluster

Abstract EPR spectra at 4, 9 and 35 GHz of hydrogenase isolated from Chromatium vinosum have been compared. The spectra at 4 and 35 GHz confirmed our earlier conclusions, made from observations at 9 GHz (Albracht, S.P.J., Kalkman, M.L. and Slater, E.C. (1983) Biochim. Biophys. Acta 724, 309–316), that the irreversibly inactivated enzyme molecules in the preparation give rise to two EPR signals due to the independent non-interacting S = 1 2 systems of Ni(III) and a |3Fe-xS| cluster. It was observed that intact enzyme molecules show a complex EPR spectrum caused by a spin-coupled pair of Ni(III) and a |4Fe-4S|3+ cluster. The interaction energy is so weak (approx. 0.01 cm−1) that the 35 GHz spectra of both the Ni(III) and the |4Fe-4S|3+ cluster have the appearance of rather normal S = 1 2 spectra with additional splittings as a result of the spin-spin interaction. At lower microwave frequencies, the spectra become increasingly complex but phenomenologically they behave as expected for an exchange-coupled pair of dissimilar ions. The distance between the two spin systems is estimated to be at the most 1.2 nm. The spin-relaxation rate of the Ni(III) ion is dramatically enhanced as a result of the coupling to the rapidly relaxing Fe-S cluster. The g values and so presumably also the ligand fields of Ni in intact and irreversibly inactivated enzyme molecules are identical. This suggests that the specific coordination of the nickel in the enzyme is not the only requirement for activity with artificial electron donors or acceptors, and that the presence of a nearby, intact |4Fe-4S|3+(3+,2+) cluster might be another essential factor. From the g values and the probable function of Ni in the enzyme we propose, as a working hypothesis, that the nickel ion has five ligands provided by the protein in a square-pyramidal coordination.

EPR evidence for direct interaction of carbon monoxide with nickel in hydrogenase from Chromatium vinosum

Abstract Exposure of reduced hydrogenase from Chromatium vinosum to carbon monoxide resulted in two new EPR-detectable nickel species, one of which was sensitive to light. Illumination of H 2 -reduced enzyme and of CO-treated enzyme caused reversible photodissociation, resulting in the same nickel species. Although the rate of this photodissociation reaction H 2 -reduced enzyme in 2 H 2 O was nearly 6-times slower than in H 2 O (Van der Zwaan, J.W., Albracht, S.P.J., Fontijn, R.D. and Slater, E.C. (1985) FEBS Lett. 179, 271–277), no difference could be observed in the case of the CO-treated enzyme. It was concluded that hydrogen, possibly as a hybride ion, and CO can bind to nickel at the same ligand position. This is in agreement with the fact that CO is a competitive inhibitor of the enzyme.

A new EPR signal of nickel in Methanobacterium thermoautotrophicum

Abstract The two main EPR signals detectable in suspensions of whole cells of Methanobacterium thermoautotrophicum strain Marburg have both been assigned to nickel by growth of the bacterium on a medium enriched in 61 Ni ( I = 3/2). One of the signals could be identified as the soluble hydrogenase, that has earlier been characterized (Albracht, S.P.J.; Graf, E.-G. and Thauer, R.K. (1982) FEBS Lett. 140, 311–313). The other signal is new. Its g values and its hyperfine structure indicate that it represents nickel in a tetragonally distorted octahedral ligand field with 4 equivalent N atoms in the equatorial plane. From the available EPR data, it could not be established whether the nickel ion is in the monovalent or trivalent state. The nickel is attached to a soluble protein. Purified methyl-coenzyme M reductase displays the same signal. The spin concentration of the species in various preparations of the enzyme accounted for at most one-fifth of the concentration of the protein-bound factor F 430 . It is proposed that the signal represents F 430 in intact, active methyl-coenzyme M reductase molecules.

Direct evidence for sulphur as a ligand to nickel in hydrogenase: an EPR study of the enzyme from Wolinella succinogenes enriched in 33S

A preparation of hydrogenase from Wolinella (formerly Vibrio) succinogenes enriched in 33S to at least 70% has been studied by EPR spectroscopy. The sulphur isotope, which has a nuclear spin of 32, clearly broadened the spectrum of the Fe-S cluster. Resolved hyperfine splitting was observed in one of the lines of the EPR spectrum of Ni(III). It was concluded that this was due to interaction with one 33S nucleus. Also the appearance of the high-field line of the EPR signal of Ni(I), either before or after photodissociation of the nickel-hydrogen bond, indicated hyperfine interaction with one 33S nucleus. No indication has been observed for nitrogen hyperfine interaction in any of the EPR spectra in this study. The data provide independent proof of earlier conclusions from EXAFS studies by two other groups (Lindahl, P.A., Kojima, N. Hausinger, R.P., Fox J.A., Teo, B.K., Walsh C.T. and Orme-Johnson, W.H. (1984) J. Am. Chem. Soc., 106, 3062–3064, and Scott R.A., Wallin, S.A., Czechowsky, M., DerVartanian, D.V., LeGall, J., Peck, H.D.,Jr. and Moura, I. (1984) J. Am. Chem. Soc. 106, 6864–6865) that sulphur is present in the direct coordination of nickel in hydrogenase. The EPR data provide evidence for the presence of only one, or possibly two, S ligands.

Destruction and reconstitution of the activity of hydrogenase from Chromatium vinosum

The specific activity and the EPR spectra of purified hydrogenase from Chromatium vinosum vary from preparation to preparation. It has been found that both properties are dependent on the redox state of specific redox groups, probably thiols, in the enzyme. In defect enzyme molecules the proposed thiol groups are oxidized to an -S-S- bridge. As a result, the activity of the enzyme in the assay with viologens is minimal and nickel and the iron-sulphur cluster in the oxidized enzyme are present as two non-interacting S = 12 systems, Ni(III) and a [3Fe-4S]1+ cluster. In intact enzyme the -S-S- bridge does not exist. Such enzyme molecules are 15- (H2-production reaction) or 65- (H2-uptake reaction) times as active as defect molecules. Intact oxidized enzyme can accomodate a spin-coupled Ni(III)/[4Fe-4S]3+ pair. It can be converted to defect molecules by partial reduction with phenazine methosulphate plus ascorbate in air at 40–50°C. The reverse transition is induced by anaerobic incubation with a disulphide-reducing agent at 50°C.

A procedure for selecting mammalian cells with an impairment in oxidative phosphorylation

The mitochondrial encephalomyopathies in man are characterized by heterogeneous defects leading to an impairment in the pathway of aerobic energy production. As a means of investigating the molecular and genetic mechanisms underlying these disorders we have developed a procedure for selecting mammalian cell lines with features resembling the human pathological phenotypes. The principle of the selection is the use of a fluorescent amphiphilic dye, 2,4-(dimethylamino)-1-styrylmethylpyridiniumiodine, a cation showing two main features. Firstly, it is accumulated by mitochondria to an extent correlated with the magnitude of the electrochemical gradient of protons across the mitochondrial inner membrane. Secondly, upon irradiation with UV light, it gives rise to formation of free radicals, which inflict damage to the cell. Mutant cells with an impairment in oxidative phosphorylation will have more chance to survive than wild type cells. The selection procedure was applied to a stock of mutagenized Chinese hamster ovary cells. After subcloning of the cells which survived the selection procedure, twenty-six independent clones were isolated. Eighteen of the clones had a partial deficiency of cytochrome c oxidase ranging from 30 to 60% of the activity in control cells. The properties of two of the clones are described. One clone has been cultured under non-selective conditions for at least 12 months with retention of the partial deficiency of cytochrome c oxidase.