Jiro Tobari

Amicyanin: An electron acceptor of methylamine dehydrogenase

Abstract A type I blue copper protein, “amicyanin”, was purified from a cell-free extract of methylamine-grown Pseudomonas AM1. It was found that amicyanin is able to serve as a primary electron acceptor of methylamine dehydrogenase. Amicyanin was reduced by the addition of both methylamine dehydrogenase and methylamine. Cytochromes c could not be directly reduced but could be reduced with the addition of amicyanin. The results strongly suggest that amicyanin participates as an electron carrier between methylamine dehydrogenase and cytochrome c in the electron transport chain of the methylamine-grown cell.

Mycobacterium smegmatis ferredoxin: A unique distribution of cysteine residues constructing iron—sulfur clusters

Some bacteria contain 8 Fe-8 S ferredoxin with two (4 Fe-4 S) clusters of widely different midpoint potentials (-700 mV apart). These ferredoxins are Azotobacter vinelandii ferredoxin I [ 11, Rhodospirikm mbnm ferredoxin HV 121 and I&cobacterium flawunz ferredoxin I( 131. EPR analyses [2-41 have shown that these ferredoxins were paramagnetic &2.01) in oxidized state and diamagnetic in reduced state, contrary to 8 Fe-S S ferredoxins from ClostUdium and C71ro-omatircnz which were paramagnetic (9= 1.94) in reduced state. This evidence shows that the two clusters function between the same pair ofoxidative states (-2 and -1 oxidation states) as the single (4 Fe-4 S) cluster in Ckomatizrn7 high-potential iron protein (HIPIP) [LB]. The :imino(~)-te:-minal sequence of fI. ninelai7dii ferredoxin I was investigated [S] and it was concluded that an unusual nature of the two clusters correlated to the different distribution of cysteine residues from that of PepPococcus ferredoxin [6]_ We have reported [7] the complete amino acid sequence of fseudon7oonas ovalis ferredoxin I which had a similar magnetic nature to that of A_ virzdmdii ferredoxin 1 and shown that the sequence was quite homologous with that of A. vinelandii ferredoxin I, but there was no substantial difference in the distribution of 8 cysteine residues between I’_ ovalis ferredoxin I and Pe~~ococcus 161 or C?1~-0991c~~~~99~ [S,S] ferredoxin. A 8 Fe-8 S ferredoxin with mol. wt 12 800 has been isolated from Mycobacteriui77 srnegnsatis Takeo [ IO] having similar properties to those ofA. vinelar7dii ferredoxjn above. This fcrredoxin contained

PARTICIPATION OF FLAVIN-ADENINE DINUCLEOTIDE IN THE ACTIVITY OF MALATE DEHYDROGENASE FROM MYCOBACTERIUM AVIUM.

Abstract 1. 1. Malate dehydrogenase was extracted from the insoluble cell fragments of Mycobacterium avium (Strain Takeo), and partially purified by ammonium sulphate fractionation. 2. 2. In particulate preparations a number of oxidants, particularly 2,6-dichlorophenolindophenol, phenazine methosulphate, and cytochrome c, serve as efficient electron-acceptors. With the extracted enzyme, a greater specificity is required in the electron-acceptor; phenazine methosulphate is the best oxidant and, to a lesser extent, ferricyanide is also active. 3. 3. Pyridine nucleotides do not participate in the action of the enzyme. The extracted dehydrogenase is activated, however, by low concentrations of FAD, but not by FMN. 4. 4. Malate dehydrogenase activity is completely inhibited by the addition of Amytal (sodium 5-ethyl-5-isoamyl-barbiturate) and heavy metals in the phenazine methosulphate assay.

Requirement of flavin adenine dinucleotide and phospholipid for the activity of malate dehydrogenase from Mycobacterium avium

Abstract In a previous communication, Kimura and Tobari (1963) described the occurence of a flavoprotein in Mycobacterium avium concerned with the dehydrogenation of malate to oxaloacetate. This is in contrast to the NAD-linked malate dehydrogenase, well documented in animal tissues and other microorganisms. Later, Asano and Brodie (1963) reported that the activity of malate-vitamine K reductase from M.phlei requires the addition of FAD. Optimal rate of activity was achieved only when vitamine K1 was suspended in phospholipid. The involvement of phospholipid has also been reported for the β-hydroxybutyrate dehydrogenase isolated from beef heart mitochondria ( Jurtshuk, Sekuzu and Green, 1961 ). In the present paper the requirement for FAD and phospholipid in order to observe the maximal activity of the soluble malate dehydrogenase purified from the particulate fraction of M.avium is described.

Isolation and characterization of a ferredoxin from Mycobacterium smegmatis takeo

Abstract A soluble ferredoxin was isolated in a crystalline form from Mycobacterium smegmatis Takeo. (This species has been identified as Mycobacterium avium strain Takeo in some previous papers. See Kusunose, M., et al. (1976) Arch. Microbiol., 108, 65–73, for the rationale for this new name.) The molecular weight was calculated to be 12035 from the amino acid sequence (Hase, T., et al. (1979) FEBS Lett., 103, 224–228). It contained 5.7 mol non-heme iron, 5.9 mol acid-labile sulfur and 8 mol cysteine residues per 12 035 g. The ferredoxin exhibited absorption maxima at 280 and 406 nm with shoulders around 330 and 450 nm, and the absorbance ratio ( A 405 A 280 ) was 0.61. The absorbance in the visible region was partially reduced by the addition of sodium dithionite, but was not affected by the addition of potassium ferricyanide. The two midpoint oxidation-reduction potentials at pH 7.0 were determined potentiometrically to be −15 and −435 mV. The EPR spectra in the isolated stateexhibited an almost isotropic signal at g 2.00, while in the dithionite-reduced state the signal was at g 2.015. Though its physiological role in the cell is unknown, the ferredoxin served as an electron mediator for cytochrome c reduction by NADPH in the presence of spinach ferredoxin-NADP+ reductase. From the data described above, M. smegmatis ferredoxin would seem to be a two FeS cluster containing ferredoxin, and at least one of the two FeS clusters is thought to be of the 3Fe3S type.

Spectroscopic and electrochemical properties of two azurins (Az-iso1 and Az-iso2) from the obligate methylotroph Methylomonas sp. strain J and the structure of novel Az-iso2.

Methylomonas sp. strain J gives rise to two azurins (Az-iso1 and Az-iso2) with methylamine dehydrogenase (MADH-Mj). The intense blue bands characteristic of Az-iso1 and Az-iso2 are observed at 621 and 616 nm in the visible absorption spectra respectively, being revealed at 620−630 nm in those of usual azurins. The EPR signal of Az-iso1, similar to usual azurins, shows axial symmetry, while the axial EPR signal of Az-iso2 involves a slightly rhombic character. The half-wave potentials (E1/2) of the two azurins and the intermolecular electron-transfer rate constants (kET) from MADH-Mj to each azurin were determined by cyclic voltammetry. The E1/2 values of Az-iso1 and Az-iso2 are +321 and +278 mV vs NHE at pH 7.0, respectively. The kET value of Az-iso2 is larger than that of Az-iso1 by a factor of 5. However, the electron-transfer rate of Az-iso2 is interestingly slower than those of the azurins from a denitrifying bacterium, Alcaligenes xylosoxidans NCIB 11015, and the amicyanin from a different methylotroph, Methylobacterium extorquens AM1. The structure of Az-iso2 has been determined and refined against 1.6 Å X-ray diffraction data. The whole structure of Az-iso2 is quite similar to those of azurins reported already. The Cu(II) site of Az-iso2 is a distorted trigonal bipyramidal geometry like those of other azurins, but some of the Cu-ligand distances and ligand-Cu-ligand bond angle parameters are slightly different. These findings suggest that Az-iso2 is a novel azurin and perhaps functions as an electron acceptor for MADH.

Mycobacterium smegmatis malate dehydrogenase: activation of the lipid-depleted enzyme by anionic phospholipids and phosphatidylethanolamine

Phospholipid-protein interactions have been investigated in a phospholipid-requiring enzyme, FAD-dependent malate dehydrogenase isolated from Mycobacterium smegmatis membranes, to correlate these interactions with enzyme function. The ability of several natural and synthetic phospholipids including CL and PE, which are major phospholipids in M. smegmatis membranes, to activate purified, lipid-depleted, enzymatically inactive malate dehydrogenase was examined. Anionic phospholipids and PE activated the enzyme, while zwitterionic phospholipids did not. A PE/PC mixture activated the enzyme in the form of both bilayer and non-bilayer structure. CL/PE mixtures activated malate dehydrogenase much more than each single phospholipid species. All anionic phospholipids used stabilized the enzyme, while PE and zwitterionic phospholipids did not. CL and a CL/PE mixture protected malate dehydrogenase from proteinase digestion, while PE did not. All phospholipids and phospholipid mixtures tested caused little secondary structural change in malate dehydrogenase. The results obtained in this study suggest that CL and CL/PE mixtures could form stable, enzymatically active complexes with malate dehydrogenase which might be similar to the native complex in M. smegmatis membranes. Although PE could activate malate dehydrogenase in both bilayer and non-bilayer form, it formed a complex with malate dehydrogenase which was inferior in terms of stability and susceptibility to proteinases, indicating that PE alone poorly reconstitutes the active enzyme-phospholipid complex.

Resonance Raman spectroscopic evidence for the presence of 4Fe and 3Fe centers in Pseudomonas ovalis ferredoxin and Mycobacterium smegmatis ferredoxin

not received Raman spectroscopy Ferredoxin 3Fe center 4Fe center Pseudomonas ovalis

Genetic organization and characterization of the mau gene cluster, which concerned the initial step of electron transport chains involved in methylamine oxidation of the obligate methylotroph Methylomonas sp. strain J

A gene cluster encoding methylamine dehydrogenase (MADH) and its primary electron acceptor, azurin iso-2 (the initial step of electron transport chains involved in methylamine oxidation) of the obligate methylotroph Methylomonas sp. strain J (Methylomonas J) was characterized. PCR products synthesized using primers based on the N- and C-terminal amino acid sequences of MADH light subunit from the facultative methylotroph Methylobacterium extorquens AM1 were used as probes for cloning. Fourteen open reading frames were found in a cloned EcoRI-PstI fragment consisting of a total of 11,549 bp. Eight open reading frames were identified as mauFBEDAGLM based on the high homology to those from Methylophilus methylotrophs W3A1-NS. The mauA and mauB genes encode L subunit and H subunit of MADH, respectively. The mauO which encodes azurin iso-2, a primary electron acceptor from MADH of Methylomonas J, was located downstream from the mauFBEDAGLM genes and the direction of transcription of this gene was found to be opposite to that of the mau gene cluster. Northern blotting analysis suggested that the expression of the mau gene cluster and the mauO gene is co-regulated. These results suggest that dynamic gene recruitment occurred for the initial step of the electron transport chains involved in methylamine oxidation of methylotrophs.

FAD-dependent malate dehydrogenase from Mycobacterium smegmatis: Activation of the lipid-depleted enzyme by incorporation into cardiolipin liposome

The lipid-depleted, enzymatically inactive malate dehydrogenase isolated from Mycobacterium smegmatis membrane was found to be incorporated spontaneously into cardiolipin liposome, but not into phosphatidylcholine liposome, as was revealed by electron spin resonance spectra with the use of 5-doxylstearic acid as a spin probe in the phospholipid liposomes. In addition, sucrose density gradient centrifugation in 0.5 M KCl showed hydrophobic interaction of the enzyme with cardiolipin liposome and further proved that the enzyme thus interacted hydrophobically with cardiolipin liposome became enzymatically active. From the results obtained above, it was concluded that the lipid-depleted, enzymatically inactive malate dehydrogenase isolated from M. smegmatis membrane was found to be activated by incorporating into the hydrophobic region of cardiolipin liposome.