A continuous-wave electron-nuclear double resonance (X-band) study of the Cu super(2+) sites of particulate methane mono-oxygenase of Methylococcus capsulatus (strain M) in membrane and pure dopamine beta -mono-oxygenase of the adrenal medulla

• Published in: Biochemical journal 2002-05, Vol.363 (3), p.677-686
• Main Authors: Katterle, B, Gvozdev, R I, Abudu, N, Ljones, T, Andersson, K K
• Format: Article
• Language: English
• ISSN: 0264-6021

All methanotrophic bacteria express a membrane-bound (particulate) methane mono-oxygenase (pMMO). In the present study, we have investigated pMMO in membrane fragments from Methylococcus capsulatus (strain M). pMMO contains a typical type-2 Cu super(2+) centre with the following EPR parameters: g sub(z) 2.24, g sub(x,y) 2.06, 19.0mT and 1.0mT. Simulation of the Cu super(2+) spectrum yielded a best match by using four equivalent nitrogens (A super(N) = 1.5mT, 42MHz). Incubation with ferricyanide neither changed nor increased the amount of EPR-active Cu super(2+), in contrast with other reports. The EPR visible copper seems not to be part of any cluster, as judged from the microwave power saturation behaviour. Continuous-wave electron-nuclear double resonance (CW ENDOR; 9.4GHz, 5-20K) experiments at g perpendicular of the Cu(II) spectrum show a weak coupling to protons with an A super(H) of 2.9MHz that corresponds to a distance of 3.8Ae (1Ae identical with 0.1nm), assuming that it is a purely dipolar coupling. Incubation in super(2)H sub(2)O leads to a significant decrease in these super(1)H-ENDOR intensities, showing that these protons are exchangeable. This result strongly suggests that the EPR visible copper site of pMMO is accessible to solvent, which was confirmed by the chelation of the Cu super(2+) by diethyldithiocarbamic acid. The super(1)H and super(14)N hyperfine coupling constants confirm a histidine ligation of the EPR visible copper site in pMMO. The hyperfine structure in the ENDOR or EPR spectra of pMMO is not influenced by the inhibitors azide, cyanide or ammonia, indicating that they do not bind to the EPR visible copper. We compared pMMO with the type-2 Cu super(2+) enzyme, dopamine beta -mono-oxygenase (D beta M). For D beta M, it is assumed that the copper site is solvent-accessible. CW ENDOR shows similar weakly coupled and super(2)H sub(2)O-exchangeable protons (2.9MHz), as observed in pMMO, as well as the strongly coupled nitrogens (40MHz) from the co-ordinating N of the histidines in D beta M. In conclusion, the resting EPR visible Cu in pMMO is not part of a trinuclear cluster, as has been suggested previously.