Inactivation of myosin subfragment one by cobalt (II) /cobalt (III) phenanthroline complexes. 2. Cobalt chelation of two critical SH groups

Wells, J.A.; Werber, M.M.; Yount, R.G.

Biochemistry 18(22): 4800-4805


ISSN/ISBN: 0006-2960
PMID: 159719
DOI: 10.1021/bi00589a006
Accession: 068522851

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Treatment of rabbit myosin chymotropic subfragment (SF1) with CoCl2, 1,10-phenanthroline (phen) and [CoIII(phen)2CO3]+ in the presence of ADP leads to parallel inactivation of the NH4+-EDTA, Ca2+ and Mg2+ ATPase activities. The incorporation of a single Co correlates with complete loss of enzyme activity. With prolonged treatment up to 2 Co per SF1 can be incorporated, indicating the existence of noncritical Co site(s). The noncritical site(s) can be prelabeled with a mixture of Co(II)- and Co-(III)-ethylenediamine-N,N'-diacetic acid complexes without affecting the ATPase activity. Subsequent labeling by Co phenanthroline complexes incorporates essentially one more Co with complete loss of enzyme activity. The loss of 2 SH groups by cobalt phenanthroline modification correlates linearly with ATPase inactivation. Selective reduction with a variety of reducing agents and subsequent removal of slightly greater than 1 cobalt allows recovery of approximately 2 SH groups and almost all ATPase activity. The loss of SH groups results from direct Co chelation as opposed to coincident disulfide formation or indirect steric blockage. Co incorporation and thiol modification with Co phenanthroline complexes can be blocked by prior treatment of SF1 with low-fold excesses of N,N'-p-phenylenedimaleimide with MgADP (believed to cross-link 2 reactive cysteines called SH1 and SH2) or N-ethylmaleimide (believed to label SH1). Pretreatment with Co phenanthroline with MgADP provides significant protection against reaction with both maleimide ragents. Co phenanthroline inactivation with MgADP probably results from the incorporation of a single Co(III) into a dithiol site consisting of SH1 and SH2. Co chelation of these SH groups means that they must move to within 3-5 .ANG. of each other. These findings, combined with the earlier 12-14-.ANG. thiol to thiol distance determined by Reisler et al., with a rigid crosslinking agent, indicate the region of myosin containing SH1 and SH2 can undergo large conformational changes.