OXYGENATION OF COBALT(II)-SUBSTITUTED LIMULUS-POLYPHEMUS HEMOCYANIN - KINETICS, CD, AND MCD STUDIES

Cobalt(II)-substituted Limulus polyphemus (CoHcy) is characterized by circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies. At neutral pH, the active site Co(II)'s are mostly aquoCoHcy, which gives rise to weak CD but intense low-temperature (4.2 K) MCD spectral features at 571, 552, and 526 nm. At higher pH's CoHcy is mostly hydroxoCoHcy and still has a weak visible CD spectrum, but three near-UV CD peaks at 372, 340, and 316 nm appear which are proposed to arise from ligand to metal charge transfer (LMCT). The 4.2 K MCD spectrum of hydroxoCoHcy is very rich with peaks at 304, 324, 355, 526, 556, 571, 616, and 642 nm. These spectra can be interpreted in terms of approximate C3, ligand symmetry about each active-site cobalt(II) with a (HiS)30 ligand set. In the case of aquoCoHcy, the 0 ligand comes from a coordinated water, whereas, in hydroxoCoHcy, the 0 ligand comes from hydroxide. The hydroxoCoHcy spectrum in the d-d transition region has too many peaks to be accounted for solely from spin-allowed transitions; therefore, it is proposed that the 616- and 642-nm bands arise from spin-forbidden 4A2 --> 2A2(G),2E(G) transitions which gain intensity from the nearby spin-allowed 4A2 --> 4E(P) and 4A2(P) transitions. The aquoCoHcy and hydroxoCoHcy MCD spectral features are strikingly similar to those of the low-pH and high-pH forms of cobalt-substituted carbonic anhydrases, respectively. HydroxyCoHcy rapidly oxygenates (k = 500 M-1 s-1) to form oxyCoHcy, which has strong CD bands at 332, 413, 518, and 618 nm. These bands are supportive of an oxyCoHcy active site, which contains mu-1,2-peroxo, mu-hydroxo dibridged Co(III) dimers. The 332- and 413-nm bands are due to pi(a)* --> d(sigma)* and pi(b)* --> d(sigma)* O2(2-) --> Co(III) LMCT, while the 518- and 618-nm CD bands are d-d transitions arising from six-coordinate Co(III).