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A few days before MF Global’s collapse, regulators stationed at the firm were assured its books were in order.

Their boss, Gary Gensler, was not convinced. A former Goldman Sachs partner who once passed the test for certified public accountants, he bore into the numbers himself and grew uneasy with the firm’s finances.

“Keep pressing them,” he told his regulators, according to people with direct knowledge of the conversation.

Within days, his suspicions were confirmed. More than $600 million in customer money was missing from MF Global, prompting an investigation by Gensler’s Commodity Futures Trading Commission.

As the regulator intensifies its inquiry into MF Global and the firm’s former leader, Jon S. Corzine, the agency must do without its hard-charging chairman. Late last week, Gensler stepped away from the investigation amid questions over his relationship to Corzine, his former boss at Goldman Sachs and a fellow Democrat.

Now, the worry is that the commission will stumble in its rare turn on center stage without its star leading the way.

Gensler’s withdrawal from the investigation came despite apparently only loose ties to Corzine. The men have seen each other just a handful of times since Gensler left Goldman Sachs in 1997. Gensler did not attend Corzine’s 2010 wedding. And Corzine did not attend the funeral of Gensler’s wife, a noted artist who died of breast cancer in 2006.

Gensler’s recusal puts the CFTC at a potential disadvantage as it embarks on its most high-profile investigation since its founding in 1974.

Under Gensler, the commission had begun to shed a reputation as a toothless watchdog, bringing a record number of enforcement cases while writing new rules to police Wall Street.

At the same time, the agency’s budget is under attack on Capitol Hill and it faces questions over its handling of MF Global. Lawyers and MF Global clients have questioned why it is taking so long to recover money that should have never gone missing at all.

“There is a huge spotlight on the agency now,” said Scott Talbott, a lobbyist for the Financial Services Roundtable, a Wall Street trade group.

Gensler has been Washington’s most aggressive ambassador to Wall Street, introducing sweeping new rules to crack down on excessive risk taking.

Even industry groups acknowledge his impact, though they are not fond of his aggressive tactics. He can be difficult, colleagues said. And his unshakeable faith in regulation has left some fearful the agency will jeopardize Wall Street’s anemic recovery and broader economic growth.

“It may be useful for Chairman Gensler in the short run to be viewed as an opponent of the financial industry, but to be successful in the long run, the CFTC will have to produce workable regulations that do not damage the economy too much,” said Steven Lofchie, a partner at Cadwalader, Wickersham & Taft. “The jury is still out on whether the CFTC can do that.”

Wall Street scourge is an unlikely addition to the resume of Gensler, 54, a marathon runner and single father of three daughters.