Specific polyisoprene-cleaving activities of 1.5 U/mg and 4.6 U/mg were determined for purified Strep-tagged latex clearing protein (Lcp) of Streptomyces sp. strain K30 at 23°C and 37°C, respectively. Metal analysis revealed the presence of approximately one atom of iron per Lcp molecule. Copper, which had been identified in Lcp1 VH2 of Gordonia polyisoprenivorans previously, was below the detection limit in Lcp K30 . Heme was identified as a cofactor in purified Lcp K30 by (i) detection of characteristic ␣-, -, and ␥ (Soret)-bands at 562 nm, 532 nm, and 430 nm in the visible spectrum after chemical reduction, (ii) detection of an acetoneextractable porphyrin molecule, (iii) determination of a heme b-type-specific absorption maximum (556 nm) after chemical conversion of the heme group to a bipyridyl-heme complex, and (iv) detection of a b-heme-specific m/z value of 616.2 via mass spectrometry. Spectroscopic analysis showed that purified Lcp as isolated contains an oxidized heme-Fe 3؉ that is free of bound dioxygen. This is in contrast to the rubber oxygenase RoxA, a c-type heme-containing polyisoprene-cleaving enzyme present in Gram-negative rubber degraders, in which the covalently bound heme firmly binds a dioxygen molecule. Lcp K30 also differed from RoxA in the lengths of the rubber degradation cleavage products and in having a higher melting point of 61.5°C (RoxA, 54.3°C). In summary, RoxA and Lcp both are equipped with a heme cofactor and catalyze an oxidative C-C cleavage reaction but differ in the heme subgroup type and in several biochemical and biophysical properties. These findings suggest differences in the catalytic reaction mechanisms.T he hydrocarbon natural rubber [poly(cis-1,4-isoprene)] is an important biopolymer that is produced by many plants and some fungi. Natural rubber, as well as chemically synthesized poly(cis-1,4-isoprene) (synthetic rubber), has been in use by mankind for more than 100 years. Huge amounts of rubber derived from the rubber tree Hevea brasiliensis are the basis for the manufacturing of tires, sealers, latex gloves, condoms, and many other items. Most of these materials are released to the environment in the form of waste or by abrasion in the case of tires. As a natural compound, rubber is a fully biodegradable material, and many rubber-degrading microorganisms have been isolated from various ecosystems in the past (1-8). The initial step of rubber biodegradation is the enzymatic cleavage of the polymeric carbon backbone to smaller products. Two types of rubber-cleaving enzymes have been characterized so far. One is rubber oxygenase A, RoxA, which is found in Gram-negative clearing zone formers. The other is latex clearing protein, Lcp, which has been identified only in Gram-positive organisms so far. RoxA first was isolated and biochemically characterized from Xanthomonas sp. strain 35Y (9). Biochemical and biophysical investigation revealed that RoxA is an extracellular dioxygenase with two covalently attached heme groups (10, 11) and is structurally but not functio...