Purification of and Kinetic Studies on a Cloned Protoporphyrinogen Oxidase from the Aerobic Bacterium Bacillus subtilis A. V. Corrigall, 1 K. B. Siziba, M. H. Maneli, E. G. Shephard, M. Ziman, T. A. Dailey,* H. A. Dailey,* R. E. Kirsch, and P. N. Meissner Lennox Eales Porphyria Laboratories, MRC/UCT Liver Research Centre, Department of Medicine, University of Cape Town Medical School, Observatory 7925, South Africa; and *Department of Microbiology and Centre for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602 Received April 20, 1998, and in revised form July 6, 1998 The previously cloned and expressed protoporphy- rinogen oxidase from Bacillus subtilis has been puri- fied to homogeneity by Ni 2 affinity chromatography using a His 6 tag and characterized. The enzyme has a molecular weight of approximately 56,000 daltons, a pI of 7.5, a pH optimum (protoporphyrinogen) of 8.7, and a noncovalently bound flavine adenine dinucleotide cofactor. The Michaelis constants (K m ) for protopor- phyrinogen-IX, coproporphyrinogen-III, and mesopor- phyrinogen-IX are 1.0, 5.29, and 4.92 M, respectively. Polyclonal antibody to B. subtilis protoporphyrinogen oxidase demonstrated weak cross-reactivity with both human and Myxococcus xanthus protoporphyrinogen oxidase. B. subtilis protoporphyrinogen oxidase is not inhibited by the diphenyl ether herbicide acifluorfen at 100 M and is weakly inhibited by methylacifluor- fen at the same concentration. Bilirubin, biliverdin, and hemin are all competitive inhibitors of this en- zyme. © 1998 Academic Press Key Words: protoporphyrinogen oxidase; Bacillus subtilis; heme biosynthesis; acifluorfen; bilirubin; biliverdin; hemin. Oxidation of protoporphyrinogen-IX to protoporphy- rin-IX, in the penultimate step in the heme biosyn- thetic pathway, is catalyzed by the enzyme protopor- phyrinogen oxidase (PPO) 2 (EC 1.3.3.4). In most eu- karyotes studied (1– 4) various forms of PPO have been shown to be mitochondrial membrane proteins, utiliz- ing molecular oxygen as an electron acceptor and flavin as a cofactor. However, recent studies on Nicotiana tabacum (where two distinct isoforms of PPO occur) (5) and Arabidopsis thaliana (4) (where a form of the pro- tein containing a targeting presequence has been de- scribed) suggest that for PPO other nonmitochondrial or premitochondrial forms of the enzyme do exist. The cell’s respiratory chain serves as electron acceptor in the prokaryote Escherichia coli (6), while the electron acceptor in the anaerobic prokaryote Desulfovibrio gi- gas is unknown (7). In the facultative anaerobe Sac- charomyces cerevisiae (8) molecular oxygen is the elec- tron acceptor under aerobic or microaerobic conditions. Four prokaryotic forms of PPO have been cloned and sequenced viz. Bacillus subtilis (9, 10), Myxococcus xanthus (6), E. coli (11), and Propionibacterium freuden- reichii (12). B. subtilis and M. xanthus PPO are similar to the eukaryotic forms studied (1, 2, 4) in that they utilize molecular oxygen and have a flavin adenine dinucleotide (FAD) binding motif at the amino termi- nus, whereas E. coli PPO does not (11). Of the PPOs studied to date, B. subtilis PPO appears to be unique in that it has a broader substrate specificity—it utilizes coproporphyrinogen-III as substrate in addition to the universal PPO substrate protoporphyrinogen-IX (10, 13). B. subtilis PPO (10), like PPO from E. coli and Bradyrhizobium japonicum (14), is not inhibited by diphenyl ethers (DPEs). This is in contrast to M. xan- thus and the eukaryotic PPOs studied which are strongly inhibited by these compounds (6, 15, 16). Defective or inhibited PPO is relevant in nature. In plants inhibition of PPO by DPEs (and some other 1 To whom correspondence should be addressed. Fax: +27 21 448 6815. E-mail: anne@liver.uct.ac.za. 2 Abbreviations used: PPO, protoporphyrinogen oxidase; FAD, fla- vin adenine dinucleotide; DPE, diphenyl ether; VP, variegate por- phyria; LB, Luria broth; NTA, nitrilotriacetic acid. 0003-9861/98 $25.00 251 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 358, No. 2, October 15, pp. 251–256, 1998 Article No. BB980834