Article Figures & Data
Figures
- FIG. 1.
SDS-PAGE of the samples obtained during purification of PCMH and content of covalently bound FAD. (A) Shown is a 12.5% polyacrylamide gel. Lanes: M, molecular mass standard; 1, membrane fraction after ultracentrifugation; 2, washed membrane fraction; 3, LDAO-treated membrane; 4, supernatant after LDAO treatment; 5, NaCl-treated membrane; 6, supernatant after NaCl treatment; 7, gel filtration pool; 8, pool after hydrophobic interaction chromatography. (B) Shown is an 8% polyacrylamide gel of purified PCMH. Lanes: M, molecular mass standard; 9, fluorescence of 1 and 1.5 μg purified PCMH (excitation at 488 nm, detection at 520 nm); 10, Coomassie blue staining of the same gel. (C) Shown is a 16.5% polyacrylamide gel of purified PCMH. Lanes: M, molecular mass standard; 11, purified PCMH. For lanes M, masses in kDa are indicated to the left.
- FIG. 2.
UV/vis spectra of PCMH (0.2 mg ml−1, pH 8.0). (A) Oxidized (straight line) and dithionite reduced (dotted line). (B) Oxidized (straight line) and p-cresol reduced (1 mM, 15 min, dotted line). (C) Oxidized (straight line) and p-hydroxybenzaldehyde reduced (1 mM, 15 min, dotted line). Insets in panels A to C show difference spectra (reduced value minus oxidized value).
- FIG. 3.
Inhibition of p-cresol oxidation by p-hydroxybenzyl alcohol. The Km values for p-cresol were determined in the presence of 0, 70, and 200 μM p-hydroxybenzyl alcohol by varying the p-cresol concentration in the presence of constant concentrations of p-hydroxybenzyl alcohol; the initial rates were was extrapolated from the regression curves for 1.7 (✸), 5.2 (•), 10.4 (⧫), 20.9 (▾), 40.0 (▴), and 120 (▪) μM p-cresol. (Left) Cornish-Bowden plot (10); (right) Dixon plot (4). Values for K ic (18 ± 9 μM) and K iu (235 ± 20 μM) were estimated from the intersection points of the regression lines.
- FIG. 4.
Model for a putative PCMH/cytochrome bc complex. (A) Proposed alternative electron transfer routes during p-cresol oxidation (dashed line) and p-hydroxybenzyl alcohol oxidation (solid line) are shown. Electrons derived from alcohol oxidation can be transferred to menaquinone, whereas those derived from p-cresol oxidation may be transferred to a periplasmic cytochrome c. Alternatively, the latter could also be transferred to menaquinone by means of energy-driven reversed electron transfer. The areas of the symbolized subunits do not represent the exact relative molecular masses. MQ, menaquinone; MQH2, menaquinol. Cyt c, cytochrome c. (B) Part of the pcm gene cluster. PCMH is encoded by pcmG (β subunit), pcmI (α subunit), and pcmJ (α′ subunit), and the putative cytochrome bc complex is encoded by pcmC (product similar to the N-terminal part of transmembrane cytochrome b), pcmD (product similar to the C-terminal part of transmembrane cytochrome b), pcmE (Rieske protein), and pcmF (cytochrome c). The function of the other putative gene products is unknown.
Tables
- TABLE 1.
Purification of PCMH from G. metallireducens
Purification step Total protein (mg) Enzymatic activityb Yield of recovery (%) Purification (fold) Total (nmol min−1) Specific (nmol min−1 mg−1) Crude extracta 819 4,750 5.8 100 1 Washed membrane 88 1,473 16.7 31 2.9 LDAO-treated membrane 66.2 417 6.3 9 1.1 NaCl supernatant 46.9 1,186 25.3 25 4.4 Gel filtration pool 9.6 988 103 21 17.8 Octyl Sepharose pool 0.4 880 2,200 18 380 - TABLE 2.
UV/vis spectral properties of PCMH
Spectral band Absorption coefficient (M−1 cm−1) Oxidized Reduced Difference α (552 nm) 20,800 42,200 21,400 β (522 nm) 26,700 30,600 3,900 Soret (410/416 nm) 182,100 (410 nm) 206,800 (416 nm) 140,400a FAD (450 nm) 48,100 24,700 23,400 ↵ a The molar absorption coefficient for the Soret is calculated from the maximum (418 nm) and the minimum (406 nm) of the difference spectrum.
- TABLE 3.
Molecular and kinetic properties of PCMH
Property Value Substrates (Km values [μM]) p-Cresol (1.7 ± 0.3); p-hydroxybenzyl alcohol (2.7 ± 0.7) Products p-Hydroxybenzyl alcohol; p-hydroxybenzaldehyde Subunit composition αα'β2; α, 57 kDa; α′, 59 kDa; β, 8.5 kDa Native mol mass 124 ± 15 kDa Cofactors One covalently linked FAD, two heme c's Absorption maxima Reduced, 416, 523, and 552 nm; oxidized, 410 and 522 nm, shoulder at 450 nm Specific activities (μmol min−1 mg−1)/turnover nos. (s−1) 2.2/4.8 (p-cresol); 0.2/0.48 (p-hydroxybenzyl alcohol) k cat/Km (mol−1 s−1) 2.98 × 106 (p-cresol); 0.20 × 106 (p-hydroxybenzyl alcohol) Inhibition constants for p-hydroxybenzyl alcohol K ic = 18 ± 9 μM; K iu = 235 ± 20 μM
